MVA-vectored universal beta-coronavirus vaccine design & development

Vaccine Insights 2023; 2(5), 201–211

DOI: 10.18609/vac.2023.032

Published: 28 June 2023
Expert Insight
M J Newman, M J Hauser, A Domi et al.

Coronaviruses capable of infecting humans have circulated within the population and are well known to the scientific community. These viruses generally cause mild-moderate and recurring respiratory infections but pose minimal serious health risks. However, the more recent emergence of SARS-CoV-1, CoV-2, and MERS clearly demonstrate the risk of new coronavirus ‘spillover events’ from animal hosts, and this risk can be addressed proactively. A significant level of antigenic variation exists for the Spike protein amongst the coronaviruses that can infect humans and include the evolving variants of SARS-CoV-2. This is a well-recognized hurdle for vaccine development where the focus is on the induction of neutralizing antibody responses. However, a significant level of sequence and antigenic similarity is also known to exist, especially for the nucleocapsid, membrane proteins, and most of the non-structural proteins, and these conserved proteins are targets of the T cell arm of the immune system. Using modern viral vector-based vaccine technologies, it is feasible to design and develop vaccines capable of inducing T cell responses specific to multiple conserved viral proteins, providing a breadth of antiviral function and specificity. Vaccines of this type could serve as the basis for better targeting both SARS-CoV-2 as well as other beta-coronaviruses in a controlled prevention manner. This type of vaccine could be used as a booster to standard-of-care products or specifically for the benefit of unique patient populations where vaccine failure is common. Critically, we could return to a focus on prophylaxis, the prevention of disease through controlled vaccine campaign strategies using products that induce durable immune responses, including immunological memory.


Limitations of first-generation vaccines for SARS-CoV-2

The response to the global COVID-19 pandemic by public health entities and the vaccine industry was unprecedented in terms of speed and resulted in the development of multiple vaccines based on different technologies. The primary design focus of the industry was on the use of the SARS-CoV-2 spike (S) protein as the vaccine immunogen with the goal of inducing high levels of neutralizing antibodies [1]Hsieh CL, Goldsmith JA, Schaub JM, et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 2020; 369, 1501–1505. [2]Polack FP, Thomas SJ, Kitchin N, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 2020; 383, 2603–2615.  [3]Ewer KJ, Barrett JR, Belij-Rammerstorfer  S, et al. T cell and antibody responses induced by a single dose of ChAdOx1 Ncov-19 (AZD1222) vaccine in a phase 1/2 clinical trial. Nat. Med. 2021; 27, 270–278.  [4]Shinde V, Bhikha S, Hoosain Z, et al. Efficacy of NVX-CoV2373 Covid-19 Vaccine Against the B.1.351 Variant. N. Engl. J. Med. 2021; 384, 1899–909. [5]Sadoff J, Gray G, Vandebosch A, et al. Safety and Efficacy of Single-Dose Ad26.Cov2.S Vaccine Against Covid-19. N. Engl. J. Med. 2021; 384, 2187–2201. [6]Voysey M, Clemens SAC, Madhi SA, et al. Safety and efficacy of the ChAdOx1 Ncov-19 Vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 2021; 397, 99–111. . By mid-2021, the combination of infection-induced and vaccine-induced antibody-positive individuals (seroprevalence) was reported to exceed 80% [7]Jones JM, Stone M, Sulaeman H, et al. Estimated US infection- and vaccine-induced SARS-CoV-2 seroprevalence based on blood donations, July 2020-May 2021. JAMA 2021; 326, 1400–1409. . This level was expected to provide a significant level of protection at the population level (herd immunity). Unfortunately, we have observed continued circulation of SARS-CoV-2 variants, making evident several limitations associated with the S-protein-focused approach of first-generation vaccines.

Of immediate concern is the emergence of immune escape variants with changes in the S-gene and -protein sequences that mediate resistance to the neutralizing capacity of vaccine-induced antibodies (Figure 1Sequence variation of the SARS-CoV-2 genome.) [8]Phillips N, The coronavirus is here to stay - Here’s what that means. Nature 2021; 590, 382–384.. This evolution of SARS-CoV-2 is driven, in part, by the continued circulation of the virus in the immune population wherein vaccine-induced antibodies can support the selection of variants that are resistant to neutralization [9]Pulliam JRC, van Schalkwyk C, Govender N, et al. Increased risk of SARS-CoV-2 reinfection associated with emergence of the Omicron variant in South Africa. medRxiv 2021; preprint. [10]Wang R, Hozumi Y, Zheng YH, Yin C, Wei GW. Host immune response driving SARS-CoV-2 evolution. Viruses 2020; 2, 1095. . Variants of concern (VOC) were recognized early in the pandemic, but it was the Delta and Omicron VOC, with almost total resistance to neutralizing antibodies, that highlighted the severity of the problem, driving new waves of infections with serious levels of morbidity [11]Garcia-Beltran WF, E. Lam EC, et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell 2021; 184, 2372–2383.e9.  [12]Wang P, Nair MS, Liu L, et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. bioRxiv 2021; preprint. [13]Planas D, Veyer D, Baidaliuk A, et al. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization. Nature 2021; 596, 276–280. [14]McCallum M, Czudnochowski N, Rosen LE, et al. Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement. bioRxiv 2021, preprint.  [15]Cao Y, Yisimayi A, Jian F, et al. BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by omicron infection. Nat 2022; 608, 593–602. [16]Suryawanshi RK, Chen IP, Ma T, et al. Limited cross-variant immunity from SARS-CoV-2 omicron without vaccination. Nature 2022; 607, 351–355.  [17]Tang J, Novak T, Hecker J, et al. Cross-reactive immunity against the SARS-CoV-2 omicron variant is low in pediatric patients with prior COVID-19 or MIS-C. Nat. Comm. 2022; 13, 2979. [18]Bowen JE, Addetia A, Dang HV, et al. Omicron spike function and neutralizing activity elicited by a comprehensive panel of vaccines. Science 2022; 377, 890–894. . To address this issue, bivalent mRNA booster vaccines based on the sequences of both the Wuhan and Omicron (BA5) were developed and received Emergency Use Authorization, accepted primarily on their ability to increase the titer and breadth of neutralizing antibody functions [19]Lin DY, Xu Y, Gu Y, et al. Effectiveness of Bivalent Boosters against Severe Omicron Infection.
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[20]Chalkias S, Harper C, Vrbicky K, et al. A bivalent Omicron-containing booster vaccine against COVID-19.
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[21]US Food and Drug Administration. Coronavirus (COVID-19) update: FDA authorizes Moderna, Pfizer-BioNTech bivalent COVID-19 vaccines for use as a booster dose (August 31, 2022). [22]Centers for Disease Control and Prevention. Stay up to date with COVID-19 vaccines including boosters. (September 8 2022).. This approach can be effective, as demonstrated in the influenza virus vaccine model, but public acceptance and compliance can be rate-limiting.

A secondary issue is that the kinetics and duration of antibody responses induced by coronavirus infection or vaccination with first-generation products are highly variable and often short-lived, thus limiting the effect of herd immunity. This variation could be a function of the immunogenicity of the S-protein, a limited helper T cell component associated with the use of only a single protein as the immunogen, undefined limitations of the vaccine platforms, an inherent issue with immune responses to coronaviruses, or any combination of these and other factors [23]Wheeler SE, Shurin GV, Yost M, et al. Differential antibody response to mRNA COVID-19 vaccines in healthy subjects. Microbiol. Spectr. 2021; 9, e00341-21.  [24]Chia WN, Zhu F, Xiang SW, et al. Dynamics of SARS-CoV-2 neutralizing antibody responses and duration of immunity: a longitudinal study. Lancet Microbe 2021; 2, e240–244.  [25]Dupont  L, Luke B, Snell LB, et al. Neutralizing antibody activity in convalescent sera from infection in humans with SARS-CoV-2 and variants of concern. Nature Micro. 2021; 6,1433–1442.  [26]Seow J, Graham C, Merrick, et al. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-C0V-2 infection in humans. Nature Micro. 2020; 5, 1598–1607.  [27]Feng C, Shi J, Fan Q, et al. Protective humoral and cellular immune responses to SARS-CoV-2 persist up to 1 year after recovery. Nat. Comm. 2021; 4984.  [28]Goel RR, Painter MM, Apostolidis SA, et al. mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern. Science 2021; 374(6572), abm0829. Antibody response durability is being addressed in the population and experimentally through the use of repeated booster immunizations and heterologous immunization strategies but more needs to be done to better define and address the existing limitations through improved vaccine design [29]Munro APS, Janani L, Cornelius V, et al. Safety and immunogenicity of seven COVID-19 vaccines as a third dose (booster) following two doses of ChAdOx1 nCov-19 or BNT162b2 in the UK (COV-BOOST): a blinded, multicentre randomised, controlled, phase 2 trial. Lancet 2021; 398, 2258–2276.  [30]Embi PJ, Levy ME, Naleway AL, et al. Effectiveness of 2-dose vaccination with mRNA COVID-19 vaccines against COVID-1-associated hospitalizations among immunocompromise adults—nine states, January-September 2021. MMWR 2021; 70,1–7..

A third issue is the often-overlooked patients with special medical limitations or needs. Within the vaccine field, this includes the part of the population that is partially immunocompromised. These individuals often cannot routinely raise nor maintain protective antibody responses following receipt of first-generation mRNA vaccines, contributing to an unacceptable level of variation in vaccine efficacy. This includes patients suffering from and/or being treated for numerous
malignancies, autoimmune disorders, transplant patients, dialysis patients, and potentially, even the aging population [31]Rugge M, Zorzi M and Guzzinati S. SARS-CoV-2 infection in the Italian Veneto region: adverse outcomes in patients with cancer. Nat. Cancer 2020; 1, 784–788.  [32]Assaad S, Avrillon V, Fournier M-L, et al. High mortality rate in cancer patients with symptoms of COVID-19 with or without detectable SARS-COV-2 on RT-PCR. Eur. J. Cancer 2020; 135, 251–259.  [33]Miyashita H, Kuno T. Prognosis of coronavirus disease 2019 (COVID-19) in patients with HIV infection in New York City. HIV Med. 2020; 22, e1–2. [34]Dai M, Liu d, Liu M, et al. Patients with cancer appear more vulnerable to SARS-COV-2: a multi-center study during the COVID-19 outbreak. Cancer Discov. 2020; 10, 783–791. [35]Shree T, Shankar, V, Lohmeyer JK, et al. CD-20-target therapy ablates de novo antibody response to vaccination but spares preestablished immunity.
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[36]Bassi J, Giannini O, Silacci-Freni C, et al. Poor neutralization and rapid decay of antibodies to SARS-CoV-2 variants in vaccinated dialysis patients. PLoS ONE 2022; 17, e0263328.  [37]Morris S, Anjan S, Pallikkuth S, et al. Reinfection with SARS-CoV-2 in solid-organ transplant recipients: incidence density and convalescent immunity prior to reinfection. Transpl. Infect Dis. 2022; 24(3), e13827.  [38]Hause AM, Baggs J, Marquez P, et al. Safety monitoring of COVD-19 mRNA vaccine booster doses among parsons aged >12 years with presumed immunocompromised status-United States. MMWR 2022; 71, 899–903. [39]Haydu JE, Maron JS, Redd, RA, et al. Humoral and cellular immunogenicity of SARS-CoV-2 vaccines in chronic lymphocytic leukemia: a prospective cohort study. Blood Adv. 2022; 6, 1671–1683.  [40]Cook, LB, O’Dell G, Vourvou E, et al. Third primary SARS-CoV-2 mRNA vaccines enhance antibody responses in most patients with hematological malignancies. Nat. Comm. 2022; 13, 6922.  [41]Reeg DB, Hogmann M, Neumann-Haefelin C, et al. SARS-CoV-2-specific T cell responses in immunocompromised individuals with cancer, HIV or solid organ transplants. Pathogens 2023; 12, 244. . Approved vaccines were generally as safe in these patients as the general population, allowing for the administration of additional booster doses, thus providing level benefit, but again this may be a limitation that can be better addressed through improved vaccine design [42]Morens DM, Taubenberger JK Fauci AS. Rethinking next-generation vaccines for coronaviruses, influenzaviruses and other respiratory viruses. Cell Host Microbe 2023; 31146–31157. .

Next-generation vaccines that will increase the magnitude, duration, and functional breadth of immune responses, including the establishment of immunological memory and responses that better protect mucosal tissues, are needed [42]Morens DM, Taubenberger JK Fauci AS. Rethinking next-generation vaccines for coronaviruses, influenzaviruses and other respiratory viruses. Cell Host Microbe 2023; 31146–31157. . The desired level of improvement must function to address risk from new VOC but also better serve the populations that are under-protected. Ideally, next-generation vaccines will also provide protection from the risk posed by the emergence or spill-over of other beta-coronaviruses, analogous to SARS-CoV-2. We believe that this can be best achieved through the design and development of vaccines that optimally engage the cellular, or T cell, arm of the immune system with epitope specificity focused on parts of the virus that are not prone to variation and immune-mediated selection of VOC.

Cellular immunity to conserved coronavirus proteins

Coronaviruses that can infect humans are large enveloped, single-stranded positive-sense RNA viruses that share a high level of sequence identity (Figure 1). The major structural proteins are S, nucleocapsid (N), envelope (E), and membrane (M) [43]Hu B, Guo H, Zhou P, Shi ZL. Characteristics of SARS-CoV-2 and COVID-19. Nat. Rev, Micro. 2021; 19, 141–154. . Numerous nonstructural proteins (NSP) and open-reading frame proteins (ORF), representing >60% of the genome, include proteins with a diverse range of activities including RNA-dependent RNA polymerase (RDRP), papain-like protease (PLpro), main protease (Mpro), helicase, exo- and endo-ribonucleases and proteins with virulence and immune system downregulation activities [44]Kindler E, Thiel V, Weber F. Interaction of SARS and MERS Coronaviruses with the antiviral interferon response.
Adv. Virus Res. 2016; 96, 219–243. 
[45]Perlman S, Netland J. Coronaviruses post-SARS: Update on replication and pathogenesis. Nature Rev. Micro. 2009; 7, 439–450. [46]Kasuga Y, Zhu B, Jang K-J & Yoo J-S. Innate immune sensing of coronavirus and viral evasion strategies. Exp. Mol. Med. 2021; 53, 723–736.  [47]Snijder EJ, Decroly E & Ziebuhr J. The nonstructural proteins directing coronavirus RNA synthesis and processing. Adv. Virus Res. 2016; 96, 59–126. . Most of the structural proteins and NSP are likely to be immunogenic, based on the prediction of T cell epitopes, and could be considered as additional vaccine targets [48]Grifoni A, Sidney J, Zhang Y, et al. A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2. Cell Host Microbe2021; 27, 671–680.e2. .

The COVID-19 pandemic spurred significant effort into the characterization of T cell response to SARS-CoV-2. As predicted, CD4+ and CD8+ T cell epitopes of structural proteins are well recognized but so are epitopes within known NSP and ORF genes [49]Grifoni A, Weiskopf D, Ramirez SI, et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell2020; 181, 489–1501.e15.  [50]Sette A, Crotty S. Pre-existing immunity to SARS-CoV-2: the knowns and unknowns. Nat. Rev. Immunol. 2020; 20, 457–458.  [51]Sette A, Crotty S. Adaptive immunity to SARS-CoV-2 and COVID-19. Cell 2021; 184, 861–880.  [52]Grifoni A, Sidney J, Vita R, et al. SARS-CoV-2 human T cell epitopes: adaptive immune response against COVID-19. Cell Host Microbe 2021; 29, 1076–1092. . Responses specific to epitopes in the S, M, and N proteins are most common, which correlates with the large size of these proteins and/or their abundance in viral particles. However, responses to epitopes in most of the NSP and ORF proteins have also been detected, indicating breath of response is common in convalescent
individuals. CD4+ T cells are prevalent, if not predominant over CD8+ T cells, following both symptomatic and asymptomatic SARS-CoV-2 infections. The majority of predicted or known T cell epitopes (85–95%) are highly conserved amongst VOC, based on amino acid sequences. [48]Grifoni A, Sidney J, Zhang Y, et al. A sequence homology and bioinformatic approach can predict candidate targets for immune responses to SARS-CoV-2. Cell Host Microbe2021; 27, 671–680.e2. .

The observation that early or pre-existing T cell responses, detected in the absence of prior documented infection and likely induced by previous infections involving coronavirus other than SARS-CoV-2, were significantly associated with lower levels of disease pathogenesis are of particular importance because this supports the belief that a significant level of protection can be mediated by T cells specific for conserved epitopes [49]Grifoni A, Weiskopf D, Ramirez SI, et al. Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals. Cell2020; 181, 489–1501.e15.  [53]Sekine T, Perez-Potti A, Rivera-Ballesteros O, et al. Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19. Cell 2020; 183, 158–168.e14.  [54]Rydyznski Moderbacher C, Ramirez SI, Dan JM, et al. Antigen-specific adaptive immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity. Cell 2020; 183, 996–1012.e19.  [55]Mateus J, Grifoni A, Tarke A, et al. Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans. Science 2020; 370, 89–94. [56]Swadling L, Diniz MO, Schmidt NM, et al. Pre-existing polymerase-specific T cells expand in abortive seronegative SARS-CoV-2. Nat. 2022; 60, 110–117.  [57]Loyal L, Braun J, Henze L, et al. Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination. Science 2021; 8; 374, eabh1823.  [58]Kundu R, Narean JS, Wang L, et al. Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts. Nat. Comm. 2022; 10; 13, 80. [59]da Silva Antunes R, Pallikkuth S, Williams E, et al. Differential T cell reactivity to endemic coronaviruses and SARS-CoV-2 in community and health care workers. J. Infect. Dis. 2021; 224, 70–80.  This idea is supported by studies completed using murine models for both SARS-CoV-1 and CoV-2, which demonstrated the critical importance of T cells, including memory T cells, for optimal protection, viral clearance and recovery [45]Perlman S, Netland J. Coronaviruses post-SARS: Update on replication and pathogenesis. Nature Rev. Micro. 2009; 7, 439–450. [60]Channappanavar R, Zhao J, Perlman S. T cell-mediated immune response to respiratory coronaviruses. Immunol. Res. 2014; 59, 118–128.  [61]Channappanavar R, Fett C, Zhao J, et al. Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome in coronavirus infection. J. Virol. 2014; 88, 11034–11044.  [62]Israelow B, Mao T, Klein J, et al. Adaptive immune determinants of viral clearance and protection in mouse models of SARS-CoV-2. Sci. Immunol. 2021; 6, eabl4509. .

The findings of both animal and human studies support the concept that the induction of T cell responses specific to conserved epitopes represents a logical approach toward the development of vaccines that can better protect against VOC. For example, an experimental mRNA vaccine based on the Wuhan sequences of S and N protected hACE2-transgenic mice against both Wuhan and Omicron virus challenges [63]Hajnik RL, Plante JA, Liang Y, et al. Dual spike and nucleocapsid mRNA vaccination confer protection against SARS-CoV-2 Omicron and Delta variants in preclinical models. Sci. Transl. Med. 2022; 14(14), eabq1945. . Evaluation of S- and N-based vaccine using the Modified Vaccinia Virus Ankara (MVA) platform protected rhesus macaques from infections with Wuhan and Delta variants and hamsters from Omicron infections [64]Routhu NK, Gangadhara S, Lai L, et al. A modified vaccinia Ankara vaccine expressing spike and nucleocapsid protects rhesus macaques against SARS-CoV-2 Delta infection. Sci. Immunol. 2022; 7, eabo0226.  [65]Wussow F, Kha M, Kim T, et al. Synthetic multiantigen MVA vaccine COH04S1 and variant-specific derivatives protect Syrian hamsters from SARS-CoV-2 Omicron subvariants. npj Vaccines 2023; 8, 41.. Thus, the importance of inducing T cell responses specific for multiple viral proteins as a focus for next-generation vaccine design cannot be underestimated.

Making a safe & effective vaccine

The production of vaccines designed to induce both CD4 and CD8 T cell responses that are broadly specific is complicated by the need to deliver multiple immunogenic proteins and to intersect multiple antigen processing and presentation pathways. Genetic vaccines, specifically nucleic acid-based vaccines and viral vectors, are best suited for this role.

Viral-vectored vaccine platforms rely on recombinant viruses engineered to express heterologous antigens to initiate pathogen-specific immune responses [66]McCann N, O’Connor D, Lambe T, et al. Viral vector vaccines. Curr. Opin. Immunol. 2022; 77, 102210.. Inherent interactions of the virus vector with the host cell stimulate innate immunity without the need for exogenous adjuvants. Immune responses induced by viral-vectored vaccines are generally characterized by a strong CD8+ T cell response, which is critical to clearance of viral infection. The most commonly used virus vectors are derived from human adenoviruses serotypes 4, 5, 26, and 35, simian adenoviruses, vesicular stomatitis virus, adeno-associated virus, poxviruses like MVA and human cytomegalovirus [67]Hanks T. New vector and vaccine platforms: mRNA, DNA, viral vectors. Curr. Opin. HIV AIDS 2022; 17, 338–344. . Each virus vector harbors a varying degree of virus replication, activation of innate immune pathways, and safety profile.

We focus on the use of the MVA vaccine vector platform. MVA is an attenuated form of vaccinia virus that was developed as a smallpox vaccine. It is a live virus vaccine that readily infects human cells, but it is replication defective and cannot produce a productive infection. It has an established safety record given its approved use as a smallpox and mpox vaccine in immunocompromised individuals [68]Pittman PR, Hahn M, Lee HS, et al. Phase 3 efficacy trial of modified vaccinia ankara as a vaccine against smallpox. N. Engl. J. Med. 2019; 381, 1897–1908.  [69]Vollmar J, Arndtz N, Eckl KM, et al. Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine. Vaccine 2006; 24, 2065–2070.. Properties of MVA that support its use as a vaccine vector for next-generation SARS-CoV-2 vaccines include:

  • MVA has a large and available genetic coding capacity allowing for the insertion of multiple genes into different sites, supporting the simultaneous expression of multiple immunogenic proteins.
  • MVA preferentially targets antigen-presenting cells in vivo, in particular cells of the dendritic cell lineage [70–72]. This is of particular importance for the induction of CD8+ T cells where antigen processing through the proteosome to generate epitopes and direct antigen presentation are required.
  • MVA also presents antigens through the cross-presentation pathway, which is highly effective for the induction of antibody and CD4+ T cell responses [73,74].
  • It lacks critical immune evasion genes present in vaccinia and allows for the induction of innate immune responses which provide an adjuvant effect [75].
  • Pre-existing immunity can impact the utility of viral vectors because antibodies block infection of target cells. This is a concern for many viral vectors, including MVA, because the majority of the world’s population born before the early 1970s were vaccinated against smallpox. However, MVA infection of cells and the subsequent expression of encoded genes does not appear to be impacted by pre-existing immunity induced by smallpox vaccination.
  • MVA can be safely and effectively used as a vaccine or a vaccine vector in people of all ages, including immunocompromised individuals [76].

These combined properties all contribute to the utility of the MVA vaccine vector system to produce next-generation coronavirus vaccines designed to induce broadly specific and functional T cell responses.

The most logical first step in the program was to produce an MVA-vectored vaccine encoding the S protein and a second structural protein. The N protein was selected because of the documented presence of T cell epitopes and positive animal model studies involving SARS-CoV-1 [77]Peng, H. Yang L-T, Wang L-y, et al. Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients. Virol. 2006; 351, 466–475.  [78]Azizi, A. Aucoin s, Tadesse H, et al. A combined nucleocapsid vaccine induces vigorous SARS-CD8+ T cell immune responses. Genet. Vaccines Ther. 2005; 3, 7–7. . The resulting vaccine, termed COH04S1 (labeled GEO-CM04S1 for clinical development by GeoVax Inc., Atlanta, GA), encodes the S and N proteins based on the Wuhan sequence. COH04S1 was extensively tested in relevant animal models and shown to induce protective immune responses characterized by T cell responses to both S and N [79]Chiuppesi F, Nguyen VH, Park Y, et al. Synthetic multiantigen MVA vaccine COH04S1 protects against SARS-CoV-2 in syrian hamsters and non-human primates. npj Vaccines 2022; 7, 7–7. [80]Chiuppesi F, Salazar M d’Alincourt, Contreras H, et al. Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nat. Comm. 2020; 11, 6121–6121.  [81]Chiuppesi F, Zaia JA, Frankel PH, et al. Safety and immunogenicity of a synthetic multiantigen modified vaccinia virus ankara-based COVID-19 vaccine (COH04S1): an open-label and randomised, phase 1 trial. Lancet Microbe 2022; 3(4), e252–e264.  [82]Chiuppesi F, Zaia JA, Faircloth K, et al. Vaccine-induced spike- and nucleocapsid-specific cellular responses maintain potent cross-reactivity to SARS-CoV-2 Delta and Omicron variants. iScience 2022; 25, 104745. Chiuppesi F, Zaia JA, Faircloth K, et al. Vaccine-induced spike- and nucleocapsid-specific cellular responses maintain potent cross-reactivity to SARS-CoV-2 Delta and Omicron variants. iScience 2022; 25, 104745. . The vaccine was successfully tested in a dose escalation safety and immunogenicity Phase 1 clinical trial, which demonstrated it to be highly effective at inducing T cell responses, both CD4 and CD8, at low vaccine doses. Importantly, and as predicted based on the studies of others, the T cell responses were not reduced when measured using Delta and Omicron-specific materials [82]Chiuppesi F, Zaia JA, Faircloth K, et al. Vaccine-induced spike- and nucleocapsid-specific cellular responses maintain potent cross-reactivity to SARS-CoV-2 Delta and Omicron variants. iScience 2022; 25, 104745. . The COH04S1 vaccine product is the initial step towards a next-generation SARS-CoV-2 vaccine with the ability to increase the breadth and durability of immune responses, and in particular to induce T cell responses to conserved epitopes in both S and N.

Phase 2 clinical trials are being run by GeoVax with a focus on different cancer treatment patients and as a booster in healthy volunteers (ClinicalTrials.gov ID: NCT04977024 and NCT04639466). The use of the vaccine in conjunction with the standard-of-care S-based vaccines, such as the currently approved bivalent mRNA vaccines, in at-risk patient populations is envisioned as the preferred market. This will include patients suffering from and/or being treated for numerous malignancies, autoimmune disorders, kidney failure and dialysis, and other conditions that compromise the immune system.

Potential for the development of a vaccine to protect beyond VOC

As noted, the MVA vaccine vector is characterized by a large genetic coding capacity that can allow the expression of multiple genes of interest and drive the expression of multiple immunogens by a single vaccine. For example, GeoVax previously designed, produced, and clinically tested a single MVA-vectored vaccine for HIV that encoded for gag, protease, reverse transcriptase, env (gp160), tat, vpu, and rev, and the human cytokine GM-CSF [83]Hellerstein M, Xu Y, Marino T, et al. Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine. Hum. Vacc. Immunother. 2021; 8, 1654–1658. . The potential for building on the MVA vaccine vector platform to produce a beta-coronavirus vaccine capable of protecting humans from future SARS-CoV-2 VOC and other circulating coronaviruses appears to be technically feasible and highly conserved NSP may be a suitable focus because of the presence of numerous CD8 T cell epitopes [84]Kared H, Redd AD, Bloch EM, et al. SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals. J. Clin. Invest. 2021; 131(5), e145476.  [85]Ferretti AP, Kula T, Wang Y, et al. Unbiased screens show CD8+ T cells of COVID-19 patients recognize shared epitopes in SARS-CoV-2 that largely reside outside the spike protein. Immunity 2020; 53, 1095–1107.e3. .

The first step in the design of a beta-coronavirus vaccine based on this approach would be the selection of NSP. There are numerous potential candidates and consideration of multiple factors needs to be included in this effort (Figure 1). We believe the priorities are as follows:

  • The level of amino sequence and T cell epitope conservation amongst different viruses needs to be significant and span across a diverse collection of viruses, beyond SARS-CoV-1 and include MERS and potentially the seasonally circulating viruses associated with the common cold. The net should be cast widely.
  • The evidence supporting the ability of T cells targeting specific proteins to contribute to the control viral replication in vivo needs to be evaluated using human epidemiology data and relevant animal models.
  • It is critical that the selected proteins do not pose a toxicity risk to the vaccinee when expressed at higher levels in vivo under the control of a vaccine vector. This includes immune system dysfunction.
  • Critical to vaccine production, the selected proteins cannot interfere with the replication of MVA in the avian cells used as the manufacturing substrate.

Based on these factors, we completed an initial analysis and found that many, but not all, of the genes in regions ORF1a and ORF1b are highly conserved and identified NSP3, NSP6, NSP12, NSP13, and NSP14 as logical targets for use as vaccine immunogens. The properties associated with these NSP are summarized in Table 1.

Table 1 NSP selected as vaccine immunogens
Protein designationImmunogenicity/antigenicity
(Selected literature citations)
Virus function/host cell interactions
NSP3Grifoni et al. [49]
Ong et al. [86]
Quadeer Ahmed McKay [87]
Grifoni et al. [52]
Protease
Type 1 interferon antagonist
NSP6Poland et al. [88]
Bacher et al. [89]
Facilitates assembly of replicase proteins
Induction of autophagosomes from host endoplasmic reticulum
Limits the expansion of phagosomes
NSP12Swadling et al. [56]
Grifoni et al. [52]
RNA-dependent RNA Polymerase (RdRp)
Replication and transcription of the entire SARS-CoV-2 genome is catalyzed by an RdRp
NSP13Le Bert et al. [90]
Swadling et al. [56]
Pan et al. [91]
Zinc binding domain in N terminus
RNA and DNA duplex unwinding with 5’ – 3’ polarity
Helicase
NSP14Mateus et al. [55]
Kared et al. [92]
Translation inhibitory factor
Inhibits host protein synthesis
Inhibits type 1 interferon viral response

The technical processes for constructing the MVA-vectored vaccines are well established and building on the existing COH04S1 or other prototype research vaccines is feasible. The cytoplasmic expression and the large capacity of MVA to stably accept foreign sequences make it a popular choice for gene delivery, especially for multi-antigen vaccines. However, like most virus-based vectors, the insertion of the foreign genes in MVA using the classic methods is laborious and time-consuming. For a multi-antigen vaccine candidate, the time taken can be multiplied by the number of inserts. Using an approach whereby the MVA genome is cloned into E. coli plasmids can significantly reduce the time required to produce new constructs and should support the expansion of MVA-vectored vaccine development [80]Chiuppesi F, Salazar M d’Alincourt, Contreras H, et al. Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform. Nat. Comm. 2020; 11, 6121–6121. .

Production of poxviruses has changed little since the 1930s and utilizes primary cells from embryonated chicken eggs. Limitations of this approach become apparent with the need for specific pathogen-free eggs and continuous introduction of cell substrate. Efforts are underway to replace primary cells with a qualified continuous cell line. Several avian cell lines have been tested that support MVA production including AGE1.CR, DF-1, and EB66 cells [93]Kallel H, Kamen AA. Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials. Biotech. J. 2015; 10, 641–747. [94]Garber DA, O’Mara LA, Zhao J, et al. Expanding the repertoire of modified vaccinia ankara-based vaccine vectors via genetic complementation strategies. PLOS One 2009. [95]Jordan I, Lohr V, Genzel Y, et al. Elements in the development of a production process for modified vaccinia virus ankara. Microorganisms 2013; 1, 100–121.. Although commercial-scale production has not been undertaken using avian cells, several vaccines were produced utilizing AGE.CR1 or DF-1 cell lines have entered clinical trials.

However, the animal testing process needed to critically evaluate candidate vaccines is complex. Testing will need to address potential adverse pathology risks associated with the NSP directly and with potential deleterious immunopathology associated with vaccine-induced T cell responses. The inclusion of infectious challenge experiments using MERS or other coronaviruses that aren’t closely related to SARS-CoV-2 will be needed. Many coronaviruses don’t utilize the ACE2 protein as the receptor for infection and this will limit the utility of the hACE2-transgenic mouse model. Reliance on mouse-adapted coronavirus models and an expanded assessment of disease pathogenesis will be required [96]Natekar JP, Pathak H, Stone S, et al. Differential pathogenesis of SARS-CoV-2 variants of concern in human ACE2-expressing mice. Viruses 2022; 14, 1139.  [97]Muñoz-Fontela C, Dowling WE, Funnell SGP, et al. Animal models for COVID-19. Nature 2020; 586(7830), 509–515.  [98]Stone S, Rothan HA, Natekar JP, et al. SARS-CoV-2 Variants of concern infect the respiratory tract and induce inflammatory response in wild-type laboratory mice. Viruses 2022; 14(1), 27.  [99]Roberts A, Deming D, Paddock CD, et al. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog. 2007; 3, 0023-0037. [100]Agnihothram S, Yount BL, Donaldson EF, et al. A mouse model for Beta-coronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. mBio 2014; 5(2).  [101]Cockrell AS, Yount BL, Scobey T, et al. A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat. Microbiol. 2016, 2(2). [102]Dinnon KH, Leist SR, Schäfer A, et al. A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures. Nature 2020; 586(7830), 560–566 [103]Kumari P, Rothan HA, Natekar JP, et al. Neuroinvasion and encephalitis following intranasal inoculation of SARS-CoV-2 in K18-hACE2 Mice. Viruses 2021; 13(1), 132.

Better preparation for the inevitable

The spillover of SARS-CoV-1 and MERS into the human population spurred short-term research interest in vaccines for pathogenic coronaviruses. Luckily, what was known and developed previously could be coupled with cutting-edge vaccine technologies for the development of efficacious first-generation SARS-CoV-2 vaccines with a rapid response mindset. However, we can safely assume future challenges from coronaviruses will evolve and as such, we must consider a more proactive focus with efforts focused on the development of next-generation vaccines. Vaccines capable of inducing durable and protective immune responses to conserved regions, with T cells as a predominant effector mechanism, are needed. The availability of such vaccines would support prophylactic vaccine strategies and campaigns, thus reducing the requirement for approaches focused on rapid response. These vaccines could be produced and distributed in a controlled and equitable manner without the stress and panic endured in the SARS-CoV-2 pandemic.

Affiliations

Mark J Newman
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

Mary J Hauser
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

Arban Domi
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

Sreenivasa Rao Oruganti
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

Pratima Kumari
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

Ashley N Zuniga
GeoVax Inc,
1900 Lake Park Drive, Ste 380
Smyrna, GA 30080

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Authorship & Conflict of Interest  

Contributions:All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.  

Acknowledgements: None.  

Disclosure and potential conflicts of interest: Newman MJ is a fulltime employee of GeoVax Inc, the licensee for the COH04S1 (also referred to as GM-CM04S1 vaccine construct described. Newman MJ and Hauser MJ are participants in the GeoVax Stock option program. A provisional patent application has been filed for the technology described in the paper. Domi A, Kumari P are fulltime employees of GeoVax Inc, the licensee for the COH04S1 (also referred to as GM-CM04S1 vaccine construct described). A provisional patent application has been filed for the technology described in the paper. Domi A, Oruganti SR, is a participant in the GeoVax Stock option program.  

Funding declaration: Domi A, Kumari P, Zuniga A received financial support for the research, authorship and/or publication of this article from GeoVax Inc.  

Article & copyright information  

Copyright: Published by Vaccine Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.  

Attribution: Copyright © 2023 GeoVax Inc. Published by Vaccine Insights under Creative Commons License Deed CC BY NC ND 4.0.  

Article source: Invited; externally peer reviewed.  

Submitted for peer review: Mar 22, 2023Revised manuscript received: Jun 6, 2023; Publication date: Jun 27, 2023.