Final Report Summary - FLUNIVAC (InFLUenza virus UNIVersal VACcine development program)
Executive Summary:
Effectively protecting the human population from seasonal and pandemic influenza has proven to be a challenge, since influenza viruses continue to escape from and evade immunity. Current influenza vaccines fail to provide long-lasting and broad protection against multiple strains of influenza. For the development of a universal influenza vaccine, we have to “do better than Nature”, since even natural influenza virus infections induce broad protective immunity to a limited extent.
FLUNIVAC aims to develop a candidate influenza vaccine based on recombinant Modified Vaccinia virus Ankara (rMVA) encoding both B-cell and T-cell response-inducing proteins, ready to commence Phase I clinical trials. Our working hypothesis is that in order to develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains of influenza virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Our approach is to target Influenza A virus proteins that i) are established vaccine antigens or have extensively been explored as potential vaccine antigens (haemagglutinin (HA), neuramidase (NA), nucleoprotein (NP), matrix-1 (M1), matrix-2 (M2)), and ii) hitherto have largely been overlooked as potential antigens, but which are relatively conserved and which would broaden the immune response (non-structural protein 1 (NS1) and the polymerase proteins (PB1, PB2, PA)). We will genetically design and optimize the targeted proteins as well as the MVA as vector in such a way that the antigens are optimally expressed and presented to appropriate cells of the immune system by antigen presenting cells of the host.
Several recombinant MVAs (rMVAs) that has been produced and tested to confirm the identity, expression and functional integrity of the influenza viral proteins under investigation, were tested in the mouse and ferret model. Based on the results from the in vitro studies and experiments using the mouse model, the eight single rMVAs and two cocktails of rMVAs were selected for evaluation in ferrets. Three MVA candidate influenza vaccines provided partial protection against lung inflammation and virus secretion. These included MVA vaccines based on M1, NA and a combination of HA-based MVA vaccines. These results are promising and pave the way for future experiments to optimize the vaccine candidates for better efficacy and broad protection.
Furthermore, the longevity of protective immunity induced with the rMVAs was assessed in ferrets, as measured by how long antibodies induced by some selected candidate vaccines persist in ferrets. The results indicated that all the vaccines induced antibodies as measured by heamagglutination inhibition titers (HAI). The antibody titers were very stable. In addition, when compared to the gold standard (adjuvanted protein antigens), the MVA candidates performed well, and moreover it was shown that adjuvanted MVA candidates might even further improve the immunogenicity of the vaccine candidates.
In order to optimize the MVA as vector to further improve its immunogenicity, five new promoter sequences were designed to optimize the expression of recombinant genes. The five MVA vector constructs were comparatively tested in mice for immunogenicity and protection in different experimental approaches. Of note, when testing prime boost vaccination strategies, all five recombinant MVAs conferred solid protection against the harsh intranasal challenge. When comparing the efficiency of single intramuscular immunizations we observed differences for the five recombinant MVA candidate vaccines. Based on this data one chimeric promoter was selected as the best new promoter system for generation of recombinant MVA vaccines.
Work on a MVA production platform based on a continuous cell line as alternative to primary chicken embryo fibroblasts, continued to focus on the development of a purification process for rMVAs to meet the regulatory requirements that a vaccine derived from a cell line must fulfill. The aim to obtain ≥ 108 IVP/mL, ≤ 10 ng cellular DNA/mL in a scalable production process on a substrate that is free of adventitious agents, in media and buffers that are free of animal-derived components, and with equipment suitable for technology transfer has been achieved.
Overall, the overall project objectives of FLUNIVAC have been largely achieved. Several interesting leads and products are in the pipeline, which justify further testing and assessing their potential to induce broad protective immunity.
Project Context and Objectives:
Effectively protecting the human population from seasonal and pandemic influenza has proven to be a challenge, since influenza viruses continue to escape from and evade immunity. Current influenza vaccines fail to provide long-lasting and broad protection against multiple strains of influenza. For the development of a universal influenza vaccine, we have to “do better than Nature”, since even natural influenza virus infections induce broad protective immunity to a limited extent.
FLUNIVAC aims to develop a candidate influenza vaccine based on recombinant Modified Vaccinia virus Ankara (rMVA) encoding both B-cell and T-cell response-inducing proteins, ready to commence Phase I clinical trials. Our working hypothesis is that in order to develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains of influenza virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Our approach is to target Influenza A virus proteins that i) are established vaccine antigens or have extensively been explored as potential vaccine antigens (haemagglutinin (HA), neuramidase (NA), nucleoprotein (NP), matrix-1 (M1), matrix-2 (M2)), and ii) hitherto have largely been overlooked as potential antigens, but which are relatively conserved and which would broaden the immune response (non-structural protein 1 (NS1) and the polymerase proteins (PB1, PB2, PA)). We will genetically design and optimize the targeted proteins as well as the MVA as vector in such a way that the antigens are optimally expressed and presented to appropriate cells of the immune system by antigen presenting cells of the host.
Therefore, FLUNIVAC has the following objectives for the 48 month duration of the project:
1. To generate rMVA that express the individual targeted influenza A proteins:
a. To generate rMVA expressing modified surface proteins HA, NA, and M2e individually for the induction of a broad virus neutralizing antibody response directed to the conserved regions of these proteins
b. To generate rMVA expressing internal proteins NP, M1, NS1, and the polymerase proteins PB1, PB2 and PA individually for the induction of broadly protective T-cell responses
c. To optimize the immune response to the abovementioned proteins by genetically modifying the genes of the targeted proteins (“modification by design”)
2. To determine the capacity of the generated individual rMVAs to induce the desired immune response in vitro and in mice
3. To evaluate selected combinations of rMVAs resulting from objective 2 for the induction of broad protective immunity in vivo
4. To assess the longevity of protective immunity induced with the rMVAs by comparison with adjuvanted vaccine preparations (‘gold-standard’) in vivo
5. To prepare for Phase I clinical trial
These objectives are complemented by the following two supportive objectives:
6. To optimize the MVA vector in terms of protein-expression kinetics and antigen delivery
7. To explore viable MVA production platforms as alternative to chicken embryo fibroblast (CEF) cells
The “modification by design” of the proteins on one hand, and of the MVA as vector on the other hand, should induce an immune response that provides better and longer-lasting protection against influenza A viruses of various subtypes than the natural variant of these proteins. Moreover, the combination of rMVA expressing optimized conserved targets for both B and T cell responses has strong potential to induce long-lasting robust cross-protective immunity. This will be tested by in vitro and in vivo assessment of the generated rMVAs in a selective procedure, as well as by comparison to adjuvanted vaccine preparations (‘gold-standard’) to assess the longevity of the immune response. In addition, we will explore the use of an avian cell line for the production of our MVA-based universal influenza vaccine candidate to improve production consistency and may help to ensure robust supplies of an affordable vaccine.
Project Results:
Influenza viruses are highly variable pathogens that cause respiratory tract infections with substantial morbidity and mortality. The high degree of variability complicates the production of effective vaccines against seasonal and pandemic influenza and there is a need for influenza vaccines that induce broadly protective immunity. In FLUNIVAC, vaccine targets were selected that are relatively conserved between various influenza viruses and that are ably to induce cross reactive antibody and T cells responses. Conserved targets for the induction of cross reactive antibody responses include the M2 protein, the neuraminidase and the stalk region of the hemagglutinin. Conserved targets for the induction of cross-reactive T cell include the internal structural proteins like the nucleoprotein (NP) and the Matrix 1 protein and the polymerases. For this project, modified vaccinia virus Ankara (MVA) was chosen as antigen delivery system, which is known for its capacity to induce antibody and T cell responses. At Erasmus MC it was hypothesized that for optimal induction of NP-specific CD8+ T cell responses it would be required to retain the NP upon expression from MVA in the cytosol of antigen presenting cells, because endogenous antigen procession is facilitated by the proteasome located in the cytosol. Therefore, MVA-NP constructs were prepared with a normal (wild type) NP and NP of which the nuclear localization signal was mutated or deleted. In addition, NP was fused to ubiquitin, to target NP directly to the proteasome for enhanced degradation. The recombinant MVA-NP constructs were used to infect antigen-presenting cells to stimulated NP specific T cells. Indeed some of the modifications resulted in enhanced T cell activation in vitro, especially of low avidity T cells and when low multiplicities of infection were used to infect the cells. However, in a mouse model for influenza virus infection, MVA expressing the wild type NP already protected mice from infection with influenza virus and the modifications could not further increase T cell responses and the protective efficacy of the MVA-NP. These findings confirmed that the induction of NP specific T cells responses is an important vaccine target, which contribute to protective immunity. A manuscript describing these findings was published in the Journal of Virology (Altenburg 2016, J. Virol.)
Also other strategies to influence antigen presentation and localization in the MVA infected cell for enhancement of immunogenicity were evaluated by partner LMU. Recombinant MVA viruses that deliver various model antigens, based on green fluorescent protein (GFP), undergoing co- or post-translational modifications to specifically target different localizations within infected cells were tested. In vitro characterization demonstrated that the GFP antigens either accumulated in the nucleus (MVA-nls-GFP), associated with cellular membrane systems (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). Upon vaccination we found significantly higher levels of GFP-specific CD8+T-cells in MVA-myr-GFP vaccinated mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8+ T cell responses to MVA-delivered target antigens.
Partner EMC also addressed the induction of antibodies directed to the stalk of HA. Again several constructs were made to redirect the antibody response from the immunodominant head region to the subdominant stalk. The modifications of HA included various ways to delete the head domain completely, hyperglycosylation of the head to shield it from recognition by specific B cells and replacement of the head by a self antigen (albumin). Although all proteins were expressed by MVA, only a few of the modified HA molecules retained a proper conformation of the stalk as was evidenced by recognition by stalk-specific antibodies that were provided by partner AIMM therapeutics. This partner has discovered antibodies that bind to the conserved influenza virus hemagglutinin (HA)-stem region using a laboratory-based cell culture method to maintain human antibody-secreting B cells. Stem specific antibodies (AT10-005 (group 1), AT10-002 (group , 2), AT10-003 (head specific) and AT10-004 (partially group 1 and fully group 2 specific) produced in stable or transient expression systems were purified and shared with partner EMC to confirm that modified HA expressed by MVA had the correct conformation.
These MVA-HA constructs were selected for further evaluation in the influenza mouse model. With some of these constructs strong stalk specific antibody could be induced, especially one of the headless constructs and the HA with a hyperglycosylated head domain. However, despite the presence of stalk specific antibodies, C57BL/6 mice were not protected from challenge infection with various influenza viruses. Apparently, stalk antibody mediated protection is not straightforward and it is possible that the stalk antibody levels were insufficiently high to afford protection. It was concluded that other vaccination regimens are required to optimize the induction of stalk specific antibody responses. Also at EMC, an important research question raised by the members of the scientific advisory board was addressed. It has been previously argued that immunity to poxviruses, induced by vaccination against smallpox or after repeated use of the vector, could intervene with poxvirus vector based vaccines. By priming mice with smallpox vaccine or empty vector we could demonstrate that the effect of pre-existing vector specific immunity of the immunogenicity of MVA-based vaccines was modest. Some effects were observed on the induction of cell-mediated immunity. However, the pre-existing immunity had no effect on the protective potential of MVA-based influenza vaccines and it was concluded that MVA is sufficiently immunogenic and protective in subjects with pre-existing immunity like individuals born before 1975 that were still vaccinated against smallpox. A paper describing these findings was accepted for publication (Altenburg et al. Scientific Reports 2018).
To assess the extent of cross-reactivity of cell mediated and antibodies induced with MVA-based influenza vaccines, serum samples and PBMC obtained in the framework of a phase I/II clinical trial with an MVA based H5 vaccine, were tested for their cross-reactivity with H5 influenza viruses belonging to antigenically distinct clades. By protein micro array, VN assay, HI and ADCC assay, MVA-vaccine induced antibodies displayed remarkable cross-reactivity. The same was true for the vaccine induced T cell responses. It was concluded, that MVA based H5 vaccine induce broad intra-subtypic immunity, which should be considered a desirable property in the light of the continuous emergence of new antigenically distinct clades if H5 viruses. A manuscript describing these results has been published recently (de Vries et al. J. Inf. Dis. 2018).
Also the immunogenic and protective potential of recombinant MVA vaccines expressing the influenza A M2 ectodomain (M2e) on the one hand and neuraminidase (NA) on the other hand was investigated by partners VIB and LMU. To examine MVA as a vaccine vector to induce immunity towards M2e, the latter was inserted in triple tandem copies in the endogenous A56 protein of MVA. To our knowledge, this is the first time to use A56 as a carrier for an exogenous antigen successfully. The sequence and the structure of the influenza M2e protein was optimized and inserted into the head-domain of the A56 protein. To this end, two MVA-M2e constructs were compared, whereby the endogenous cysteine-residues present in M2e were either retained or mutated to serine. This comparison was done to see whether these residues might have an influence on the structure and immunogenicity of the A56-M2e chimeric protein, since A56 itself is structured by the presence of cysteine-residues. What these experiments showed, was that retaining the cysteine-residues did influence the immunogenicity of the construct in a negative manner, lowering antibody responses. Mutating these residues to serine created a highly immunogenic vaccine, which induced a strong antibody response able to convey clear protection against influenza A. A further dissection of the immune response showed that on the one hand antibodies, in conjunction with Fcγ-receptors, are responsible for conveying this protection. On the other hand, M2e-specific T cells, which can be induced by MVA-M2e vaccination, played a subsidiary, but influential, role in MVA-M2e based protection. Thus, the main technology result is the functionality of the newly designed and constructed fusion antigens using the MVA A56 protein.
Recombinant MVAs expressing NA were designed and tested in vivo. Both type 1 (N1) and type 2 (N2) neuraminidases were used. MVA-NA vaccines were able to induce clear anti-NA antibody responses, able of recognizing both recombinant NA as well as viral NA. More importantly, these antibodies can inhibit the activity of het NA enzyme, an important feature for an NA-based vaccine candidate. Inhibition of NA activity has been stipulated as being an important correlate of protection against influenza A virus infection in human. Infection of MVA-NA immunized mice revealed a strong protection, with almost no weight loss and a very significant reduction in virus replication in the lungs. Mechanistically, transfer of antibodies to naive mice also led to transfer of immunity, without the need for engagement of Fcγ-receptors, implying inhibition of NA activity by antibodies is the major protective mechanism involved. Various vaccine candidates that showed promise in the mouse model were also tested in ferrets by partner Artemis. In two series of experiments, individual and combinations of recombinant MVA were tested in ferrets and their protective efficacy against challenge infection with H5N1 or pandemic H1N1 virus evaluated. Three MVA candidate influenza vaccines provided partial protection against lung inflammation and virus secretion in this animal model. These included MVA vaccines expressing HAwt, NA or a combination of hyperglycosylated HA and HA without the head domain. These results are promising and warrant further studies to optimize the vaccine candidates for better efficacy and broad protection.
In addition, an experiment was conducted to study how long antibodies induced by some selected candidate vaccines persist in ferrets. To this end, groups of 6 ferrets were vaccinated twice with different MVA vaccines, as follows: 1. MVA-HAwt; 2. MVA-HA gly ; 3. HAwt; 4. HA gly; 5. HA +M1 adjuvant; 6. HA gly +M1 adjuvant. Blood was collected from the animals at regular time points. The results indicated that all the vaccines induced antibodies as measured by heamagglutination inhibition titers (HAI). The antibody titers were very stable and did not wane for up to 100 days.
To improve the overall performance of the MVA vector system MVA-specific transcriptional regulation of foreign gene sequences inserted into the MVA genome was investigated by LMU. The main objective of these studies was to optimize MVA-specific gene expression for the production of influenza virus antigens. First we designed and generated various chimeric MVA promoter systems using DNA synthesis technologies. Then, we identified the most promising new promoter sequences characterizing the expression of recombinant genes within the MVA life cycle. In the FLUNIVAC program we succeeded to test five different MVA recombinant vaccines using new promoter systems, namely Psyn LMU1, PsynLMU2, PsynLMU3, PsynLMU4 and Pvgf. To test the vaccine efficiency we used the influenza virus nucleoprotein (NP) as antigen and completed the quality control experiments confirming the genetic purity and stability of all vector viruses. In in vivo experiments we characterized the immunogenicity and protective capacity of MVA-NP candidate vaccines based on these MVA vector vaccines. Based on this data we could select the promoter system PsynLMU4 as the most promising new promoter developed in FLUNIVAC. In consequence, we cloned also the influenza virus hemagglutinin (HA) protein under the control of PsynLMU4 and demonstrated the genetic purity and stability of the viruses and the preclinical in vivo characterization of recombinant MVA-PsynLMU4-HA in the mouse model. The main technology result is the development of new promoter constructs combining different stages of viral life cycle. A major achievement is the identification and selection of the PLMU4 promoter system for antigen delivery to afford robust immunogenicity and protective capacity.
Another important approach to improve MVA as viral vector was to get more information about the type and the levels of recombinant gene expression in MVA infected cells. Interestingly, in cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant MVA produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of early viral gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines.
For the analysis of vaccine induced antibody responses in mice and ferrets AIMM focused on expanding the B cell culture technology to non-human species. Long-term culture of mouse B cells is difficult and cumbersome when using hybridoma or single cell sorting and sequencing technologies. However, when using the AIMSelect technology, which is based on the retro- and/or lentiviral transduction of B cells with the BCL6 and Bcl-xL transgenes, it was possible to grow and select antigen-specific mouse B cells. Although the B cell selection (FACS sorting) and growth conditions were not as robust compared to human or rabbit B cells, relatively high levels of pure B cell subpopulations (derived from Spleen, bone marrow and lymph nodes) were obtained using cell separation methods other than by Flow Cytometry. Furthermore, novel mixtures of general stimuli and cytokines to obtain fast growing mouse B cells that could be transduced with BCL6 and Bcl-xL (transduction frequencies ~70-80%) were developed. These B cells could be cloned at the single cell level and expressed the surface immunoglobulin and secrete antibody into the culture supernatant.
The mouse B cell culture method was first implemented on samples that AIMM received from partner Novavax. B cells were cultured from frozen splenocytes obtained from mice vaccinated with HA-protein or MVA-HA based vaccines in the absence or presence of M1-adjuvant; blood was taken 14 days after the last boost. Antibodies present in the culture supernatant of the mouse B cell culture were tested for binding to influenza virus A/PR8 , a group 1 virus) and PR8/C6 chimeer proteins (obtained from Erasmus MC and F. Krammer). Although IgG+ B cell numbers were generally low, mice that received MVA-HAglyc+M1 or MVA-HAwt developed head specific antibodies but not in the MVA-HAglyc vaccinated animals. Interestingly 5 stem-reactive B cell clones were detected in the HAglyc protein vaccinated group. Four of these clones were broadly reactive with other group 1 and group 2 viruses and also one clone from the MVA-HAglyc+M1 vaccinated group.
Subsequently, mouse splenocytes were analyzed obtained from mice vaccinated with MVA expressing modified HA. However, significant number of B cells producing stem-specific antibodies could not be detected despite the detection of stem specific serum antibodies (against the PR8/C6 chimeer).
The immunogenicity of MVA-based vaccines was compared to that of vaccines based on recombinant proteins, produced by partner Q-Biologicals, with or without an adjuvant by partner Novavax. To this end, the immune response to wild type hemagglutinin (HAwt), mutated HA, (HAglyc) wild type neuraminidase (NA) and wild type nucleoprotein (NP) formulated with and without saponin-based Matrix-M adjuvant was compared to that of the corresponding recombinant (r)MVA-based vaccines in mice. In addition, the effect of the adjuvant Matrix-M on the immune response by different MVA-based vaccines was investigated.
The immunogenicity, antibody- and cell-mediated, of all tested proteins was significantly enhanced by Matrix-M adjuvant compared to protein alone. In general, the immunogenicity of adjuvanted protein preparations was comparable to that of MVA-based vaccines. Of interest, rMVA-based HAglyc vaccine induced more HA-stem specific antibodies than the rMVA-HAwt vaccine. In contrast, adjuvanted HAwt and HAglyc protein vaccines induced similar levels of HA-stem specific antibodies, and at higher levels than by rMVA-HAglyc, implying that use of Matrix-M adjuvant could help direct the immune system towards the HA stem region regardless of HA head glycosylation. Of special interest, the use of Matrix-M also improved the immunogenicity of the rMVA-HAwt, leading to higher antibody responses.
To obtain a better understanding of the mechanisms underlying the adjuvant effect of Marix-M on protein- and MVA-induced immune responses, the early immune responses in the draining lymph nodes were investigated, which are thought to be important for strong antibody and T cell responses. The total cell count per dLN of mice vaccinated with Matrix-M-adjuvanted HA, or rMVA-HA, showed more than a two-fold increase compared to the vaccines without adjuvant. Although not as strong as for the adjuvanted vaccines, vaccination with unadjuvanted rMVA-HA also resulted in an increase in total cell count 48 hours after vaccination. For all vaccines tested, CD4+ T lymphocytes comprised the largest proportion of the total cell population, followed by CD8+ T and B lymphocytes. Mice vaccinated with adjuvanted HA or rMVA-HA, and to a lesser extent unadjuvanted rMVA-HA, showed a strong increase in proportion and total number of monocytes in the dLN over time, indicating improved recruitment and/or proliferation of these cells.
The most important conclusion from this work is that Matrix-M adjuvanted recombinant influenza virus proteins are good bench mark vaccines, with respect to induction of early-, antibody- and cell-mediated immunity, for the rMVA-based influenza vaccines evaluated in the project.
Current influenza vaccines are produced at the manufacturing scale by propagating the virus in embryonated chicken eggs. Although being well established, large-scale production in embryonated eggs has limitations in terms of production scale capacity and time required for manufacture. Conventionally, MVA-based vaccines are propagated in primary chicken embryo fibroblasts (CEF), which would suffer from similar disadvantages. Therefore, partner ProBioGen AG developed an alternative production platform for recombinant MVAs, which is not dependent on primary cells but instead makes use of its proprietary avian cell line AGE1.CR.pIX.
The advantages of using a continuous cell line are obvious and lie in the scalability of the process by using bioreactors at the desired volume. Currently, up to 2000 L bioreactors, theoretically able to provide several millions of vaccine doses per batch are commercially available. Furthermore, production conditions can be tightly monitored and controlled in this environment and production times as well as potential failures can be reduced. However, since regulatory requirements for vaccines produced from continuous cell lines are much more stringent compared to that of classical CEF cells, extended efforts for subsequent purification of MVA had to be addressed.
Within the FLUNIVAC program, the AGE1.CR.pIX cells as substrate for MVA propagation were characterized extensively and robust upstream and downstream production processes were established which were well suitable for providing material for the intended preclinical studies and future scale up. Because, a large fraction of infectious MVA virus particles remained cell-associated, complementary chemically-defined media formulations were developed: a cell proliferation medium for single-cell cultivation in routine maintenance and expansion of CR.pIX cells in stirred-tank as well as in wave bioreactors, and a virus production medium that is added at the time of infection. The virus production medium induces cell aggregates that we hypothesize should allow efficient replication of viruses with a tendency to spread by direct cell-to-cell contact. This process is robust and consistently gave reliable yields beyond 108 pfu of MVA/mL at cell densities between 0.5 × 106 and 2.5 × 106 cells/mL and MOI between 0.01 and 0.1 (Jordan et al., Microorganisms 2013, 1, 100-121)
Over time, the MVA production based on AGE1.CR.pIX cells could be further improved and can be conducted in an efficient and stable manner using AGE1.CR.pIX cells in a two-stage semi-continuous and in continuous stirred tank cultivation systems (Tapia et al., PLoS One. 2017 Aug 24;12(8)). More recently, we could demonstrate that the MVA titers can be increased even further by using high-cell-density cultivation of AGE1.CR.pIX cells, a process that requires both fed-batch and medium exchange strategies (Vázquez-Ramírez et al., Vaccine. 2018 Feb 9).
In addition, we addressed the question regarding the genetic stability of MVA during passaging in AGE1.CR.pIX cells. Though rMVAs are well known for exceptional genetic stability, recombination within the MVA genome can occur, which might affect the stability of the gene of interest. To examine the stability of the recombinant MVAs expressing either NP, HA or GFP (provided by the consortium) within AGE1.CR.pIX cells we infected these cultures in chemically defined media at MOI of 0.05 using these three rMVAs. Subsequently, an uninfected culture has been infected with cryogenic or sonicated lysates of the previous infection. This procedure has been repeated for a total of 20 passages. Samples of each passage have been kept, DNA has been isolated and PCRs have been performed against the six deletion sites (see Kremer et al., 2012) to validate the integrity of the transgene (located in deletion site III). In all cases these experiments revealed no loss of the transgene, even after the 20th passage. Retrospectively, the titer of all passages was quantified that remained constant during the period of this passaging experiment leading to values in the range of 1x108 – 1x109 PFU/ml. Finally, we sequenced the transgenes of the recombinant MVAs after the 20th passage and could attest 100% accordance with the original sequences.
A fully scalable and reproducible downstream process to purify MVA-based vaccines was established. During manufacturing, intracellular MVA is released from cells by a shear-based disruption method and a primary filtration step is performed to clear cellular debris. Subsequently, chromatographic pseudo-affinity capture through a weak cationic ion exchanger removes major impurities including about 96% of host cell proteins and 99.9% of contaminating cellular DNA. Further purification and concentration is achieved by a consecutive tangential flow filtration based method, which in parallel allows the transfer of the material into a formulation buffer compatible with intramuscular injection. The final product fulfils preclinical and regulatory requirements in regard to virus titer (≥ 1x108 Infectious Units/ml), DNA load (≤ 10 ng cellular DNA/Dose) and host cell protein contaminants (>95% reduction throughout the process). Although the overall process yields of about 25% can probably be further improved, the process as it stands will be sufficient to provide material for several hundred thousand or even millions of vaccine doses.
Potential Impact:
Despite the availability of effective influenza vaccines, each year up to 650 000 people die from infections and a greater number still are hospitalized with severe morbidity after infection (www.cdc.gov/flu; Brammer et al., Influenza Other Respi Viruses, 2009). Current licensed influenza vaccines provide only short-lived strain-specific protection due to the constant antigenic drift of the virus, with the added impairment that a mismatch between vaccine and circulating strain can occur (Jong et al., JMedVir, 2000; Flannery et al., MMWR, 2015). Furthermore, influenza viruses also undergo antigenic shift, whereby a novel influenza virus enters the human population. As such there is little or no pre-existing immunity resulting in wide-spread infection, morbidity and mortality. Clearly there is a need to develop vaccines with a broader antigenic scope, for improved influenza vaccines that can induce broadly protective and long lasting immune responses to various influenza viruses.Within the FLUNIVAC project, the major goal was formulating a combinatorial influenza vaccine based on MVA as a vaccine vector. The different research groups involved explored different B cell and T cell antigens derived from influenza virus in their capacity to induce protective immunity against influenza A virus infection.
The work performed in this project entailed two aspects. On the one hand the performance of the vector under various conditions was tested. Full understanding of the advantages and limitations of the MVA antigen delivery system is of great importance for further acceptance of this vaccine platform in the future. Specifically, we have demonstrated that the presence of pre-existing poxvirus specific immunity does not affect the induction of protective immunity by MVA immunization.
The other aspect deals with the use of selected conserved influenza viral target for the induction of broadly protective immunity. By definition conserved proteins are regions thereof need to be used, because these contain epitopes shared by various influenza viruses recognized by virus specific antibodies and T cells.
The different experiments performed clearly indicated the MVA-vaccines under investigation are highly immunogenic and can induce a protective level of antibodies. Previous research has clearly shown the potential benefit of M2e- and NA-specific immunity in protection against influenza infection, and this in both preclinical and clinical settings (Schotsaert et al, Exp Rev Vaccines, 2009; Eichelberger & Wan, Curr Top Microbiol Immmunol, 2015). Moreover, although immunity towards M2e and NA is not neutralizing, it has been shown to be broad in protection. Hence, including an M2e- and/or NA-based MVA-vectored vaccine component in a combinatorial influenza vaccine could have a wide societal impact by broadening the protective efficacy towards drifted and shifted influenza viruses which enter the human populations causing influenza epidemics and pandemics.
Furthermore, it was shown that the use of the viral nucleoprotein induces cross-reactive CD8+ T cell responses that can afford protection against severe disease in mice. Modifications of NP to enhance antigen processing and presentation resulted in increased T cell activation in vitro, but did not improve the performance of MVA-NP vaccines in vivo. This approach may still be viable for the induction of T cell responses to subdominant epitopes and should be further explored. The results obtained with MVA expressing modified HA molecules were somewhat disappointing. Although with some of these constructs stalk specific antibodies could be induced, the levels were insufficient to afford protection against challenge infection. It was concluded that alternative vaccination regimens may be required to further boost the stalk specific antibody levels in order to achieve protection against infection. In contrast, the human antibody response induced with a MVA-H5 vaccine displayed a high degree of cross-reactivity with a large variety of H5 strains belonging to various antigenically distinct clades as was demonstrated in multiple serological assays. Also the MVA-H5 induced T cell response was cross-reactive with various H5 viruses. Based on these findings it was concluded that MVA is a safe and promising vaccine platform with which strong intra subtypic cross-reactive antibodies can be achieved. Although not truly “universal”, it is already a major improvement over conventional inactivated vaccine preparations that induce narrow strains specific antibody responses.
The results indicate that rationale development of a broadly protective vaccine is possible. The societal implications are huge since such a vaccine would eliminate the need for annual vaccination of the risk groups. Furthermore, the risk of vaccine failure due to un-matching vaccine would be lower.
Several MVA vector vaccines developed within FLUNIVAC were shown to have the potential to improve immunogenicity and protective capacity of MVA vaccination. Importantly, the new approaches for modified antigen presentation (A56-fusion antigens) and the new promoter systems (P_LMU4) are likely to have a major impact to enhance the intrinsic efficacy of MVA vaccines and to contribute to the successful development of a broadly protective MVA-FLUNIVAC vaccine.
The stem specific antibodies can potentially be used as a prophylactic drug to prevent serious influenza induced disease. Although the out-come of clinical trials with stem specific antibodies do not (yet) have resulted in the selection of any candidates for clinical development, these antibodies still hold promise. To help ongoing influenza research these antibodies can be used as a tool to guide structure-based-vaccine-design, similar to how they were used during this study. Regarding the mouse B cell immortalization method that we have optimized; this is very useful for academic or commercial parties that want to develop novel antibodies that have been generated in mice after vaccination. These antibodies can then be used for research, diagnostic or clinical purposes.
The production and purification of MVA utilizing cell substrates independent of primary chicken embryo fibroblasts (i) reduces the risk of a supply shortfall, (ii) leads to high MVA titers, (iii) allows the usage of chemically defined media to minimize risks to vaccine recipients and to improve lot-to-lot consistency and (iii) offers high cell-density vaccine production processes including continuous processes, so that greater volumes of affordable injectable vaccine preparations can be obtained using a fully documented production cell line.
Additionally, the work performed in FLUNIVAC has shown that it is possible to significantly improve the immunogenicity of recombinantly produced influenza protein vaccines, making them equally immunogenic to rMVA-based vaccines, suggesting that adjuvanted recombinant proteins could be a good influenza vaccine alternative. We could also show antigen-dose sparing effects when formulating influenza proteins with Matrix-M adjuvant, which may be a key factor to produce large quantities of influenza vaccine at a reasonable cost. The main point addressed by FLUNIVAC is one of today’s largest unmet medical needs, an universal influenza vaccine that protects against seasonal, avian and pandemic influenza strains in humans. This point has mainly been addressed by designing different rMVA vaccines with the goal to eliminate the need for yearly updates of seasonal influenza vaccines and contribute to pandemic preparedness. We have shown that the strong immunogenicity induced by these rMVA-based influenza vaccines can be even further improved by formulation with Matrix-M adjuvant. Thus, adjuvanted rMVA-based influenza vaccines could contribute even further to the goal of producing influenza vaccines with longer-lasting and broader protection against multiple strains of influenza virus.
The results within the FLUNIVAC project may contribute to better and more cost-effective influenza vaccines that in the long run can lead to reduced influenza infection morbidity and reduced medical care costs. This would have a great positive impact on society and be socio-economically beneficial.
List of Websites:
www.flunivac.eu
Effectively protecting the human population from seasonal and pandemic influenza has proven to be a challenge, since influenza viruses continue to escape from and evade immunity. Current influenza vaccines fail to provide long-lasting and broad protection against multiple strains of influenza. For the development of a universal influenza vaccine, we have to “do better than Nature”, since even natural influenza virus infections induce broad protective immunity to a limited extent.
FLUNIVAC aims to develop a candidate influenza vaccine based on recombinant Modified Vaccinia virus Ankara (rMVA) encoding both B-cell and T-cell response-inducing proteins, ready to commence Phase I clinical trials. Our working hypothesis is that in order to develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains of influenza virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Our approach is to target Influenza A virus proteins that i) are established vaccine antigens or have extensively been explored as potential vaccine antigens (haemagglutinin (HA), neuramidase (NA), nucleoprotein (NP), matrix-1 (M1), matrix-2 (M2)), and ii) hitherto have largely been overlooked as potential antigens, but which are relatively conserved and which would broaden the immune response (non-structural protein 1 (NS1) and the polymerase proteins (PB1, PB2, PA)). We will genetically design and optimize the targeted proteins as well as the MVA as vector in such a way that the antigens are optimally expressed and presented to appropriate cells of the immune system by antigen presenting cells of the host.
Several recombinant MVAs (rMVAs) that has been produced and tested to confirm the identity, expression and functional integrity of the influenza viral proteins under investigation, were tested in the mouse and ferret model. Based on the results from the in vitro studies and experiments using the mouse model, the eight single rMVAs and two cocktails of rMVAs were selected for evaluation in ferrets. Three MVA candidate influenza vaccines provided partial protection against lung inflammation and virus secretion. These included MVA vaccines based on M1, NA and a combination of HA-based MVA vaccines. These results are promising and pave the way for future experiments to optimize the vaccine candidates for better efficacy and broad protection.
Furthermore, the longevity of protective immunity induced with the rMVAs was assessed in ferrets, as measured by how long antibodies induced by some selected candidate vaccines persist in ferrets. The results indicated that all the vaccines induced antibodies as measured by heamagglutination inhibition titers (HAI). The antibody titers were very stable. In addition, when compared to the gold standard (adjuvanted protein antigens), the MVA candidates performed well, and moreover it was shown that adjuvanted MVA candidates might even further improve the immunogenicity of the vaccine candidates.
In order to optimize the MVA as vector to further improve its immunogenicity, five new promoter sequences were designed to optimize the expression of recombinant genes. The five MVA vector constructs were comparatively tested in mice for immunogenicity and protection in different experimental approaches. Of note, when testing prime boost vaccination strategies, all five recombinant MVAs conferred solid protection against the harsh intranasal challenge. When comparing the efficiency of single intramuscular immunizations we observed differences for the five recombinant MVA candidate vaccines. Based on this data one chimeric promoter was selected as the best new promoter system for generation of recombinant MVA vaccines.
Work on a MVA production platform based on a continuous cell line as alternative to primary chicken embryo fibroblasts, continued to focus on the development of a purification process for rMVAs to meet the regulatory requirements that a vaccine derived from a cell line must fulfill. The aim to obtain ≥ 108 IVP/mL, ≤ 10 ng cellular DNA/mL in a scalable production process on a substrate that is free of adventitious agents, in media and buffers that are free of animal-derived components, and with equipment suitable for technology transfer has been achieved.
Overall, the overall project objectives of FLUNIVAC have been largely achieved. Several interesting leads and products are in the pipeline, which justify further testing and assessing their potential to induce broad protective immunity.
Project Context and Objectives:
Effectively protecting the human population from seasonal and pandemic influenza has proven to be a challenge, since influenza viruses continue to escape from and evade immunity. Current influenza vaccines fail to provide long-lasting and broad protection against multiple strains of influenza. For the development of a universal influenza vaccine, we have to “do better than Nature”, since even natural influenza virus infections induce broad protective immunity to a limited extent.
FLUNIVAC aims to develop a candidate influenza vaccine based on recombinant Modified Vaccinia virus Ankara (rMVA) encoding both B-cell and T-cell response-inducing proteins, ready to commence Phase I clinical trials. Our working hypothesis is that in order to develop a universal influenza vaccine that provides longer-lasting and broader protection against multiple strains of influenza virus both broad-reactive antibody (B-cell) and T-cell responses need to be induced. Our approach is to target Influenza A virus proteins that i) are established vaccine antigens or have extensively been explored as potential vaccine antigens (haemagglutinin (HA), neuramidase (NA), nucleoprotein (NP), matrix-1 (M1), matrix-2 (M2)), and ii) hitherto have largely been overlooked as potential antigens, but which are relatively conserved and which would broaden the immune response (non-structural protein 1 (NS1) and the polymerase proteins (PB1, PB2, PA)). We will genetically design and optimize the targeted proteins as well as the MVA as vector in such a way that the antigens are optimally expressed and presented to appropriate cells of the immune system by antigen presenting cells of the host.
Therefore, FLUNIVAC has the following objectives for the 48 month duration of the project:
1. To generate rMVA that express the individual targeted influenza A proteins:
a. To generate rMVA expressing modified surface proteins HA, NA, and M2e individually for the induction of a broad virus neutralizing antibody response directed to the conserved regions of these proteins
b. To generate rMVA expressing internal proteins NP, M1, NS1, and the polymerase proteins PB1, PB2 and PA individually for the induction of broadly protective T-cell responses
c. To optimize the immune response to the abovementioned proteins by genetically modifying the genes of the targeted proteins (“modification by design”)
2. To determine the capacity of the generated individual rMVAs to induce the desired immune response in vitro and in mice
3. To evaluate selected combinations of rMVAs resulting from objective 2 for the induction of broad protective immunity in vivo
4. To assess the longevity of protective immunity induced with the rMVAs by comparison with adjuvanted vaccine preparations (‘gold-standard’) in vivo
5. To prepare for Phase I clinical trial
These objectives are complemented by the following two supportive objectives:
6. To optimize the MVA vector in terms of protein-expression kinetics and antigen delivery
7. To explore viable MVA production platforms as alternative to chicken embryo fibroblast (CEF) cells
The “modification by design” of the proteins on one hand, and of the MVA as vector on the other hand, should induce an immune response that provides better and longer-lasting protection against influenza A viruses of various subtypes than the natural variant of these proteins. Moreover, the combination of rMVA expressing optimized conserved targets for both B and T cell responses has strong potential to induce long-lasting robust cross-protective immunity. This will be tested by in vitro and in vivo assessment of the generated rMVAs in a selective procedure, as well as by comparison to adjuvanted vaccine preparations (‘gold-standard’) to assess the longevity of the immune response. In addition, we will explore the use of an avian cell line for the production of our MVA-based universal influenza vaccine candidate to improve production consistency and may help to ensure robust supplies of an affordable vaccine.
Project Results:
Influenza viruses are highly variable pathogens that cause respiratory tract infections with substantial morbidity and mortality. The high degree of variability complicates the production of effective vaccines against seasonal and pandemic influenza and there is a need for influenza vaccines that induce broadly protective immunity. In FLUNIVAC, vaccine targets were selected that are relatively conserved between various influenza viruses and that are ably to induce cross reactive antibody and T cells responses. Conserved targets for the induction of cross reactive antibody responses include the M2 protein, the neuraminidase and the stalk region of the hemagglutinin. Conserved targets for the induction of cross-reactive T cell include the internal structural proteins like the nucleoprotein (NP) and the Matrix 1 protein and the polymerases. For this project, modified vaccinia virus Ankara (MVA) was chosen as antigen delivery system, which is known for its capacity to induce antibody and T cell responses. At Erasmus MC it was hypothesized that for optimal induction of NP-specific CD8+ T cell responses it would be required to retain the NP upon expression from MVA in the cytosol of antigen presenting cells, because endogenous antigen procession is facilitated by the proteasome located in the cytosol. Therefore, MVA-NP constructs were prepared with a normal (wild type) NP and NP of which the nuclear localization signal was mutated or deleted. In addition, NP was fused to ubiquitin, to target NP directly to the proteasome for enhanced degradation. The recombinant MVA-NP constructs were used to infect antigen-presenting cells to stimulated NP specific T cells. Indeed some of the modifications resulted in enhanced T cell activation in vitro, especially of low avidity T cells and when low multiplicities of infection were used to infect the cells. However, in a mouse model for influenza virus infection, MVA expressing the wild type NP already protected mice from infection with influenza virus and the modifications could not further increase T cell responses and the protective efficacy of the MVA-NP. These findings confirmed that the induction of NP specific T cells responses is an important vaccine target, which contribute to protective immunity. A manuscript describing these findings was published in the Journal of Virology (Altenburg 2016, J. Virol.)
Also other strategies to influence antigen presentation and localization in the MVA infected cell for enhancement of immunogenicity were evaluated by partner LMU. Recombinant MVA viruses that deliver various model antigens, based on green fluorescent protein (GFP), undergoing co- or post-translational modifications to specifically target different localizations within infected cells were tested. In vitro characterization demonstrated that the GFP antigens either accumulated in the nucleus (MVA-nls-GFP), associated with cellular membrane systems (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). Upon vaccination we found significantly higher levels of GFP-specific CD8+T-cells in MVA-myr-GFP vaccinated mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8+ T cell responses to MVA-delivered target antigens.
Partner EMC also addressed the induction of antibodies directed to the stalk of HA. Again several constructs were made to redirect the antibody response from the immunodominant head region to the subdominant stalk. The modifications of HA included various ways to delete the head domain completely, hyperglycosylation of the head to shield it from recognition by specific B cells and replacement of the head by a self antigen (albumin). Although all proteins were expressed by MVA, only a few of the modified HA molecules retained a proper conformation of the stalk as was evidenced by recognition by stalk-specific antibodies that were provided by partner AIMM therapeutics. This partner has discovered antibodies that bind to the conserved influenza virus hemagglutinin (HA)-stem region using a laboratory-based cell culture method to maintain human antibody-secreting B cells. Stem specific antibodies (AT10-005 (group 1), AT10-002 (group , 2), AT10-003 (head specific) and AT10-004 (partially group 1 and fully group 2 specific) produced in stable or transient expression systems were purified and shared with partner EMC to confirm that modified HA expressed by MVA had the correct conformation.
These MVA-HA constructs were selected for further evaluation in the influenza mouse model. With some of these constructs strong stalk specific antibody could be induced, especially one of the headless constructs and the HA with a hyperglycosylated head domain. However, despite the presence of stalk specific antibodies, C57BL/6 mice were not protected from challenge infection with various influenza viruses. Apparently, stalk antibody mediated protection is not straightforward and it is possible that the stalk antibody levels were insufficiently high to afford protection. It was concluded that other vaccination regimens are required to optimize the induction of stalk specific antibody responses. Also at EMC, an important research question raised by the members of the scientific advisory board was addressed. It has been previously argued that immunity to poxviruses, induced by vaccination against smallpox or after repeated use of the vector, could intervene with poxvirus vector based vaccines. By priming mice with smallpox vaccine or empty vector we could demonstrate that the effect of pre-existing vector specific immunity of the immunogenicity of MVA-based vaccines was modest. Some effects were observed on the induction of cell-mediated immunity. However, the pre-existing immunity had no effect on the protective potential of MVA-based influenza vaccines and it was concluded that MVA is sufficiently immunogenic and protective in subjects with pre-existing immunity like individuals born before 1975 that were still vaccinated against smallpox. A paper describing these findings was accepted for publication (Altenburg et al. Scientific Reports 2018).
To assess the extent of cross-reactivity of cell mediated and antibodies induced with MVA-based influenza vaccines, serum samples and PBMC obtained in the framework of a phase I/II clinical trial with an MVA based H5 vaccine, were tested for their cross-reactivity with H5 influenza viruses belonging to antigenically distinct clades. By protein micro array, VN assay, HI and ADCC assay, MVA-vaccine induced antibodies displayed remarkable cross-reactivity. The same was true for the vaccine induced T cell responses. It was concluded, that MVA based H5 vaccine induce broad intra-subtypic immunity, which should be considered a desirable property in the light of the continuous emergence of new antigenically distinct clades if H5 viruses. A manuscript describing these results has been published recently (de Vries et al. J. Inf. Dis. 2018).
Also the immunogenic and protective potential of recombinant MVA vaccines expressing the influenza A M2 ectodomain (M2e) on the one hand and neuraminidase (NA) on the other hand was investigated by partners VIB and LMU. To examine MVA as a vaccine vector to induce immunity towards M2e, the latter was inserted in triple tandem copies in the endogenous A56 protein of MVA. To our knowledge, this is the first time to use A56 as a carrier for an exogenous antigen successfully. The sequence and the structure of the influenza M2e protein was optimized and inserted into the head-domain of the A56 protein. To this end, two MVA-M2e constructs were compared, whereby the endogenous cysteine-residues present in M2e were either retained or mutated to serine. This comparison was done to see whether these residues might have an influence on the structure and immunogenicity of the A56-M2e chimeric protein, since A56 itself is structured by the presence of cysteine-residues. What these experiments showed, was that retaining the cysteine-residues did influence the immunogenicity of the construct in a negative manner, lowering antibody responses. Mutating these residues to serine created a highly immunogenic vaccine, which induced a strong antibody response able to convey clear protection against influenza A. A further dissection of the immune response showed that on the one hand antibodies, in conjunction with Fcγ-receptors, are responsible for conveying this protection. On the other hand, M2e-specific T cells, which can be induced by MVA-M2e vaccination, played a subsidiary, but influential, role in MVA-M2e based protection. Thus, the main technology result is the functionality of the newly designed and constructed fusion antigens using the MVA A56 protein.
Recombinant MVAs expressing NA were designed and tested in vivo. Both type 1 (N1) and type 2 (N2) neuraminidases were used. MVA-NA vaccines were able to induce clear anti-NA antibody responses, able of recognizing both recombinant NA as well as viral NA. More importantly, these antibodies can inhibit the activity of het NA enzyme, an important feature for an NA-based vaccine candidate. Inhibition of NA activity has been stipulated as being an important correlate of protection against influenza A virus infection in human. Infection of MVA-NA immunized mice revealed a strong protection, with almost no weight loss and a very significant reduction in virus replication in the lungs. Mechanistically, transfer of antibodies to naive mice also led to transfer of immunity, without the need for engagement of Fcγ-receptors, implying inhibition of NA activity by antibodies is the major protective mechanism involved. Various vaccine candidates that showed promise in the mouse model were also tested in ferrets by partner Artemis. In two series of experiments, individual and combinations of recombinant MVA were tested in ferrets and their protective efficacy against challenge infection with H5N1 or pandemic H1N1 virus evaluated. Three MVA candidate influenza vaccines provided partial protection against lung inflammation and virus secretion in this animal model. These included MVA vaccines expressing HAwt, NA or a combination of hyperglycosylated HA and HA without the head domain. These results are promising and warrant further studies to optimize the vaccine candidates for better efficacy and broad protection.
In addition, an experiment was conducted to study how long antibodies induced by some selected candidate vaccines persist in ferrets. To this end, groups of 6 ferrets were vaccinated twice with different MVA vaccines, as follows: 1. MVA-HAwt; 2. MVA-HA gly ; 3. HAwt; 4. HA gly; 5. HA +M1 adjuvant; 6. HA gly +M1 adjuvant. Blood was collected from the animals at regular time points. The results indicated that all the vaccines induced antibodies as measured by heamagglutination inhibition titers (HAI). The antibody titers were very stable and did not wane for up to 100 days.
To improve the overall performance of the MVA vector system MVA-specific transcriptional regulation of foreign gene sequences inserted into the MVA genome was investigated by LMU. The main objective of these studies was to optimize MVA-specific gene expression for the production of influenza virus antigens. First we designed and generated various chimeric MVA promoter systems using DNA synthesis technologies. Then, we identified the most promising new promoter sequences characterizing the expression of recombinant genes within the MVA life cycle. In the FLUNIVAC program we succeeded to test five different MVA recombinant vaccines using new promoter systems, namely Psyn LMU1, PsynLMU2, PsynLMU3, PsynLMU4 and Pvgf. To test the vaccine efficiency we used the influenza virus nucleoprotein (NP) as antigen and completed the quality control experiments confirming the genetic purity and stability of all vector viruses. In in vivo experiments we characterized the immunogenicity and protective capacity of MVA-NP candidate vaccines based on these MVA vector vaccines. Based on this data we could select the promoter system PsynLMU4 as the most promising new promoter developed in FLUNIVAC. In consequence, we cloned also the influenza virus hemagglutinin (HA) protein under the control of PsynLMU4 and demonstrated the genetic purity and stability of the viruses and the preclinical in vivo characterization of recombinant MVA-PsynLMU4-HA in the mouse model. The main technology result is the development of new promoter constructs combining different stages of viral life cycle. A major achievement is the identification and selection of the PLMU4 promoter system for antigen delivery to afford robust immunogenicity and protective capacity.
Another important approach to improve MVA as viral vector was to get more information about the type and the levels of recombinant gene expression in MVA infected cells. Interestingly, in cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant MVA produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of early viral gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines.
For the analysis of vaccine induced antibody responses in mice and ferrets AIMM focused on expanding the B cell culture technology to non-human species. Long-term culture of mouse B cells is difficult and cumbersome when using hybridoma or single cell sorting and sequencing technologies. However, when using the AIMSelect technology, which is based on the retro- and/or lentiviral transduction of B cells with the BCL6 and Bcl-xL transgenes, it was possible to grow and select antigen-specific mouse B cells. Although the B cell selection (FACS sorting) and growth conditions were not as robust compared to human or rabbit B cells, relatively high levels of pure B cell subpopulations (derived from Spleen, bone marrow and lymph nodes) were obtained using cell separation methods other than by Flow Cytometry. Furthermore, novel mixtures of general stimuli and cytokines to obtain fast growing mouse B cells that could be transduced with BCL6 and Bcl-xL (transduction frequencies ~70-80%) were developed. These B cells could be cloned at the single cell level and expressed the surface immunoglobulin and secrete antibody into the culture supernatant.
The mouse B cell culture method was first implemented on samples that AIMM received from partner Novavax. B cells were cultured from frozen splenocytes obtained from mice vaccinated with HA-protein or MVA-HA based vaccines in the absence or presence of M1-adjuvant; blood was taken 14 days after the last boost. Antibodies present in the culture supernatant of the mouse B cell culture were tested for binding to influenza virus A/PR8 , a group 1 virus) and PR8/C6 chimeer proteins (obtained from Erasmus MC and F. Krammer). Although IgG+ B cell numbers were generally low, mice that received MVA-HAglyc+M1 or MVA-HAwt developed head specific antibodies but not in the MVA-HAglyc vaccinated animals. Interestingly 5 stem-reactive B cell clones were detected in the HAglyc protein vaccinated group. Four of these clones were broadly reactive with other group 1 and group 2 viruses and also one clone from the MVA-HAglyc+M1 vaccinated group.
Subsequently, mouse splenocytes were analyzed obtained from mice vaccinated with MVA expressing modified HA. However, significant number of B cells producing stem-specific antibodies could not be detected despite the detection of stem specific serum antibodies (against the PR8/C6 chimeer).
The immunogenicity of MVA-based vaccines was compared to that of vaccines based on recombinant proteins, produced by partner Q-Biologicals, with or without an adjuvant by partner Novavax. To this end, the immune response to wild type hemagglutinin (HAwt), mutated HA, (HAglyc) wild type neuraminidase (NA) and wild type nucleoprotein (NP) formulated with and without saponin-based Matrix-M adjuvant was compared to that of the corresponding recombinant (r)MVA-based vaccines in mice. In addition, the effect of the adjuvant Matrix-M on the immune response by different MVA-based vaccines was investigated.
The immunogenicity, antibody- and cell-mediated, of all tested proteins was significantly enhanced by Matrix-M adjuvant compared to protein alone. In general, the immunogenicity of adjuvanted protein preparations was comparable to that of MVA-based vaccines. Of interest, rMVA-based HAglyc vaccine induced more HA-stem specific antibodies than the rMVA-HAwt vaccine. In contrast, adjuvanted HAwt and HAglyc protein vaccines induced similar levels of HA-stem specific antibodies, and at higher levels than by rMVA-HAglyc, implying that use of Matrix-M adjuvant could help direct the immune system towards the HA stem region regardless of HA head glycosylation. Of special interest, the use of Matrix-M also improved the immunogenicity of the rMVA-HAwt, leading to higher antibody responses.
To obtain a better understanding of the mechanisms underlying the adjuvant effect of Marix-M on protein- and MVA-induced immune responses, the early immune responses in the draining lymph nodes were investigated, which are thought to be important for strong antibody and T cell responses. The total cell count per dLN of mice vaccinated with Matrix-M-adjuvanted HA, or rMVA-HA, showed more than a two-fold increase compared to the vaccines without adjuvant. Although not as strong as for the adjuvanted vaccines, vaccination with unadjuvanted rMVA-HA also resulted in an increase in total cell count 48 hours after vaccination. For all vaccines tested, CD4+ T lymphocytes comprised the largest proportion of the total cell population, followed by CD8+ T and B lymphocytes. Mice vaccinated with adjuvanted HA or rMVA-HA, and to a lesser extent unadjuvanted rMVA-HA, showed a strong increase in proportion and total number of monocytes in the dLN over time, indicating improved recruitment and/or proliferation of these cells.
The most important conclusion from this work is that Matrix-M adjuvanted recombinant influenza virus proteins are good bench mark vaccines, with respect to induction of early-, antibody- and cell-mediated immunity, for the rMVA-based influenza vaccines evaluated in the project.
Current influenza vaccines are produced at the manufacturing scale by propagating the virus in embryonated chicken eggs. Although being well established, large-scale production in embryonated eggs has limitations in terms of production scale capacity and time required for manufacture. Conventionally, MVA-based vaccines are propagated in primary chicken embryo fibroblasts (CEF), which would suffer from similar disadvantages. Therefore, partner ProBioGen AG developed an alternative production platform for recombinant MVAs, which is not dependent on primary cells but instead makes use of its proprietary avian cell line AGE1.CR.pIX.
The advantages of using a continuous cell line are obvious and lie in the scalability of the process by using bioreactors at the desired volume. Currently, up to 2000 L bioreactors, theoretically able to provide several millions of vaccine doses per batch are commercially available. Furthermore, production conditions can be tightly monitored and controlled in this environment and production times as well as potential failures can be reduced. However, since regulatory requirements for vaccines produced from continuous cell lines are much more stringent compared to that of classical CEF cells, extended efforts for subsequent purification of MVA had to be addressed.
Within the FLUNIVAC program, the AGE1.CR.pIX cells as substrate for MVA propagation were characterized extensively and robust upstream and downstream production processes were established which were well suitable for providing material for the intended preclinical studies and future scale up. Because, a large fraction of infectious MVA virus particles remained cell-associated, complementary chemically-defined media formulations were developed: a cell proliferation medium for single-cell cultivation in routine maintenance and expansion of CR.pIX cells in stirred-tank as well as in wave bioreactors, and a virus production medium that is added at the time of infection. The virus production medium induces cell aggregates that we hypothesize should allow efficient replication of viruses with a tendency to spread by direct cell-to-cell contact. This process is robust and consistently gave reliable yields beyond 108 pfu of MVA/mL at cell densities between 0.5 × 106 and 2.5 × 106 cells/mL and MOI between 0.01 and 0.1 (Jordan et al., Microorganisms 2013, 1, 100-121)
Over time, the MVA production based on AGE1.CR.pIX cells could be further improved and can be conducted in an efficient and stable manner using AGE1.CR.pIX cells in a two-stage semi-continuous and in continuous stirred tank cultivation systems (Tapia et al., PLoS One. 2017 Aug 24;12(8)). More recently, we could demonstrate that the MVA titers can be increased even further by using high-cell-density cultivation of AGE1.CR.pIX cells, a process that requires both fed-batch and medium exchange strategies (Vázquez-Ramírez et al., Vaccine. 2018 Feb 9).
In addition, we addressed the question regarding the genetic stability of MVA during passaging in AGE1.CR.pIX cells. Though rMVAs are well known for exceptional genetic stability, recombination within the MVA genome can occur, which might affect the stability of the gene of interest. To examine the stability of the recombinant MVAs expressing either NP, HA or GFP (provided by the consortium) within AGE1.CR.pIX cells we infected these cultures in chemically defined media at MOI of 0.05 using these three rMVAs. Subsequently, an uninfected culture has been infected with cryogenic or sonicated lysates of the previous infection. This procedure has been repeated for a total of 20 passages. Samples of each passage have been kept, DNA has been isolated and PCRs have been performed against the six deletion sites (see Kremer et al., 2012) to validate the integrity of the transgene (located in deletion site III). In all cases these experiments revealed no loss of the transgene, even after the 20th passage. Retrospectively, the titer of all passages was quantified that remained constant during the period of this passaging experiment leading to values in the range of 1x108 – 1x109 PFU/ml. Finally, we sequenced the transgenes of the recombinant MVAs after the 20th passage and could attest 100% accordance with the original sequences.
A fully scalable and reproducible downstream process to purify MVA-based vaccines was established. During manufacturing, intracellular MVA is released from cells by a shear-based disruption method and a primary filtration step is performed to clear cellular debris. Subsequently, chromatographic pseudo-affinity capture through a weak cationic ion exchanger removes major impurities including about 96% of host cell proteins and 99.9% of contaminating cellular DNA. Further purification and concentration is achieved by a consecutive tangential flow filtration based method, which in parallel allows the transfer of the material into a formulation buffer compatible with intramuscular injection. The final product fulfils preclinical and regulatory requirements in regard to virus titer (≥ 1x108 Infectious Units/ml), DNA load (≤ 10 ng cellular DNA/Dose) and host cell protein contaminants (>95% reduction throughout the process). Although the overall process yields of about 25% can probably be further improved, the process as it stands will be sufficient to provide material for several hundred thousand or even millions of vaccine doses.
Potential Impact:
Despite the availability of effective influenza vaccines, each year up to 650 000 people die from infections and a greater number still are hospitalized with severe morbidity after infection (www.cdc.gov/flu; Brammer et al., Influenza Other Respi Viruses, 2009). Current licensed influenza vaccines provide only short-lived strain-specific protection due to the constant antigenic drift of the virus, with the added impairment that a mismatch between vaccine and circulating strain can occur (Jong et al., JMedVir, 2000; Flannery et al., MMWR, 2015). Furthermore, influenza viruses also undergo antigenic shift, whereby a novel influenza virus enters the human population. As such there is little or no pre-existing immunity resulting in wide-spread infection, morbidity and mortality. Clearly there is a need to develop vaccines with a broader antigenic scope, for improved influenza vaccines that can induce broadly protective and long lasting immune responses to various influenza viruses.Within the FLUNIVAC project, the major goal was formulating a combinatorial influenza vaccine based on MVA as a vaccine vector. The different research groups involved explored different B cell and T cell antigens derived from influenza virus in their capacity to induce protective immunity against influenza A virus infection.
The work performed in this project entailed two aspects. On the one hand the performance of the vector under various conditions was tested. Full understanding of the advantages and limitations of the MVA antigen delivery system is of great importance for further acceptance of this vaccine platform in the future. Specifically, we have demonstrated that the presence of pre-existing poxvirus specific immunity does not affect the induction of protective immunity by MVA immunization.
The other aspect deals with the use of selected conserved influenza viral target for the induction of broadly protective immunity. By definition conserved proteins are regions thereof need to be used, because these contain epitopes shared by various influenza viruses recognized by virus specific antibodies and T cells.
The different experiments performed clearly indicated the MVA-vaccines under investigation are highly immunogenic and can induce a protective level of antibodies. Previous research has clearly shown the potential benefit of M2e- and NA-specific immunity in protection against influenza infection, and this in both preclinical and clinical settings (Schotsaert et al, Exp Rev Vaccines, 2009; Eichelberger & Wan, Curr Top Microbiol Immmunol, 2015). Moreover, although immunity towards M2e and NA is not neutralizing, it has been shown to be broad in protection. Hence, including an M2e- and/or NA-based MVA-vectored vaccine component in a combinatorial influenza vaccine could have a wide societal impact by broadening the protective efficacy towards drifted and shifted influenza viruses which enter the human populations causing influenza epidemics and pandemics.
Furthermore, it was shown that the use of the viral nucleoprotein induces cross-reactive CD8+ T cell responses that can afford protection against severe disease in mice. Modifications of NP to enhance antigen processing and presentation resulted in increased T cell activation in vitro, but did not improve the performance of MVA-NP vaccines in vivo. This approach may still be viable for the induction of T cell responses to subdominant epitopes and should be further explored. The results obtained with MVA expressing modified HA molecules were somewhat disappointing. Although with some of these constructs stalk specific antibodies could be induced, the levels were insufficient to afford protection against challenge infection. It was concluded that alternative vaccination regimens may be required to further boost the stalk specific antibody levels in order to achieve protection against infection. In contrast, the human antibody response induced with a MVA-H5 vaccine displayed a high degree of cross-reactivity with a large variety of H5 strains belonging to various antigenically distinct clades as was demonstrated in multiple serological assays. Also the MVA-H5 induced T cell response was cross-reactive with various H5 viruses. Based on these findings it was concluded that MVA is a safe and promising vaccine platform with which strong intra subtypic cross-reactive antibodies can be achieved. Although not truly “universal”, it is already a major improvement over conventional inactivated vaccine preparations that induce narrow strains specific antibody responses.
The results indicate that rationale development of a broadly protective vaccine is possible. The societal implications are huge since such a vaccine would eliminate the need for annual vaccination of the risk groups. Furthermore, the risk of vaccine failure due to un-matching vaccine would be lower.
Several MVA vector vaccines developed within FLUNIVAC were shown to have the potential to improve immunogenicity and protective capacity of MVA vaccination. Importantly, the new approaches for modified antigen presentation (A56-fusion antigens) and the new promoter systems (P_LMU4) are likely to have a major impact to enhance the intrinsic efficacy of MVA vaccines and to contribute to the successful development of a broadly protective MVA-FLUNIVAC vaccine.
The stem specific antibodies can potentially be used as a prophylactic drug to prevent serious influenza induced disease. Although the out-come of clinical trials with stem specific antibodies do not (yet) have resulted in the selection of any candidates for clinical development, these antibodies still hold promise. To help ongoing influenza research these antibodies can be used as a tool to guide structure-based-vaccine-design, similar to how they were used during this study. Regarding the mouse B cell immortalization method that we have optimized; this is very useful for academic or commercial parties that want to develop novel antibodies that have been generated in mice after vaccination. These antibodies can then be used for research, diagnostic or clinical purposes.
The production and purification of MVA utilizing cell substrates independent of primary chicken embryo fibroblasts (i) reduces the risk of a supply shortfall, (ii) leads to high MVA titers, (iii) allows the usage of chemically defined media to minimize risks to vaccine recipients and to improve lot-to-lot consistency and (iii) offers high cell-density vaccine production processes including continuous processes, so that greater volumes of affordable injectable vaccine preparations can be obtained using a fully documented production cell line.
Additionally, the work performed in FLUNIVAC has shown that it is possible to significantly improve the immunogenicity of recombinantly produced influenza protein vaccines, making them equally immunogenic to rMVA-based vaccines, suggesting that adjuvanted recombinant proteins could be a good influenza vaccine alternative. We could also show antigen-dose sparing effects when formulating influenza proteins with Matrix-M adjuvant, which may be a key factor to produce large quantities of influenza vaccine at a reasonable cost. The main point addressed by FLUNIVAC is one of today’s largest unmet medical needs, an universal influenza vaccine that protects against seasonal, avian and pandemic influenza strains in humans. This point has mainly been addressed by designing different rMVA vaccines with the goal to eliminate the need for yearly updates of seasonal influenza vaccines and contribute to pandemic preparedness. We have shown that the strong immunogenicity induced by these rMVA-based influenza vaccines can be even further improved by formulation with Matrix-M adjuvant. Thus, adjuvanted rMVA-based influenza vaccines could contribute even further to the goal of producing influenza vaccines with longer-lasting and broader protection against multiple strains of influenza virus.
The results within the FLUNIVAC project may contribute to better and more cost-effective influenza vaccines that in the long run can lead to reduced influenza infection morbidity and reduced medical care costs. This would have a great positive impact on society and be socio-economically beneficial.
List of Websites:
www.flunivac.eu