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An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces protective immuni

Issuing time:2021-10-21 10:30

Abstract

Early in the SARS-CoV-2 pandemic concerns were raised regarding infection of new animal hosts and the effect on viral epidemiology. Infection of other animals could be detrimental by causing clinical disease, allowing further mutations, and bares the risk for the establishment of a non-human reservoir. Cats were the first reported animals susceptible to natural and experimental infection with SARS-CoV-2. Given the concerns these findings raised, and the close contact between humans and cats, we aimed to develop a vaccine candidate that could reduce SARS-CoV-2 infection and in addition to prevent spread among cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus, known to be safe and efficacious in a variety of animal species, could induce neutralizing antibody responses in guinea pigs and cats. The design of the SARS-CoV-2 spike immunogen was critical in developing a strong neutralizing antibody response. Vaccination of cats was able to induce high neutralizing antibody responses, effective also against the SARS-CoV-2 B.1.1.7 variant. Interestingly, in contrast to control animals, the infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after SARS-CoV-2 challenge. Correspondingly, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit the virus. The results show that the RP vaccine induces protective immunity preventing SARS-CoV-2 infection and transmission. These data suggest that this RP vaccine could be a multi-species vaccine useful to prevent infection and spread to and between animals should that approach be required.

Introduction

SARS-CoV-2 is an extremely contagious respiratory coronavirus that emerged in China in late 2019 and has since spread globally causing the on-going coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses are enveloped, single-stranded, non-segmented, positive-sense RNA viruses that encode sixteen non-structural proteins and four structural proteins. The structural Spike (S) protein is the major determinant of host cell tropism by binding to the angiotensin-converting enzyme 2 (ACE2) on cells, a type I integral membrane protein that plays an important role in human vascular health. Using the ACE2 receptor to gain entry to cells in the upper respiratory tract (URT), SARS-CoV-2 infection of humans has manifested itself in a wide range of clinical outcomes, from asymptomatic to very severe respiratory infections which in some situations are complicated by immunological dysfunction causing COVID-19 with over 3,4 million fatalities to date. As ACE2 orthologs that are highly similar to the human ACE2 receptor are also present on the cells of a number of other animals it is important to understand whether those potential hosts can play any role in disease spread. Since the first human infections, it has been shown that cats, dogs, ferrets, hamsters, and mink can be readily infected either in laboratory studies or via natural transmission1,2,3,4,5. The role these susceptible animals play in the human epidemiology is unclear, though two-way transmission between mink and humans has been demonstrated leading to the culling of all animals in mink farms from Denmark and the Netherlands3. It is therefore of utmost importance to understand the role of animals in the spread of this virus, especially with regard to their potential to act as a viral reservoir and to develop important viral variants which thereby influence the overall epidemiology.

The importance of cats in the epidemiology of COVID-19 has yet to be fully established, though there are a significant number of reports of cats testing positive for SARS-CoV-2, mostly in association with human infections in the same household. The first published report demonstrating that cats could be experimentally infected also showed virus transmission to in-contact cats1. Whilst the infected cats did not demonstrate overt clinical disease, significant respiratory lesions were detected post-mortem especially in younger cats. In subsequent experimental trials, no clinical disease was observed in challenged cats, but prolonged shed of virus and spread to contact cats was again detected 45. In addition to these experimental infection studies, there have been numerous reports of domestic cats testing positive for SARS-CoV-2, with less than a quarter showing signs of disease and no severe presentations as reported in humans6. Although a number of these cases were associated with the presence of a confirmed SARS-CoV-2 infected owner, this was not always the case7, and natural infection between domestic cats has not been ruled out. Serological surveys of cats in China7, USA8, France9, and Italy10 have demonstrated that a high proportion of cats tested positive for SARS-CoV-2, including feral animals with no known history of ownership. Although concerns regarding feline infections have significantly reduced, the initial reports that a large number of cats were being abandoned by owners11 led to key opinion leaders releasing statements regarding the low risk of human infection from cats6. Furthermore, owners testing positive for SARS-CoV-2 have been advised to distance themselves from their cats in an attempt to prevent transmission, and SARS-CoV-2 infection of cats is now reportable to the World Organization for Animal Health (historically, OIE)12. Recently, with the rise of new variants, there are also reports that these variants may have altered host tropism13 and possibly different pathogenesis14. For these reasons it is important to further study the epidemiology of SARS-CoV-2 in cats, and whether the possibility exists of the feline population becoming a natural reservoir for the virus.

A large number of human vaccines are now in development against SARS-CoV-2, with more than ten approved for use in various regions globally under an Emergency Use Authorization (EUA). The types of vaccine include adjuvanted expressed SARS-CoV-2 S protein, adjuvanted whole SARS-CoV-2 virus vaccines, mRNA encoding SARS-CoV-2 S, and recombinant viral vector vaccines expressing the SARS-CoV-2 S15. Early reports indicate that these vaccines have good safety profiles and have greatly reduced both the number and severity of infections. Other important considerations for the long term success of these vaccines include the immunological correlates of the protection induced, the vaccination scheme required to induce an appropriate duration of immunity, protection against variant strains, the cost and production scale of these vaccines required for the global population as well as storage and transportation temperatures, potential rare side effects such as vaccine-induced thrombotic thrombocytopenia16 and whether any antibody-dependent enhancement is detected, as has been seen on rare occasions with other coronavirus vaccine candidates17. The use of coronavirus vaccines in the veterinary industry is well established with a variety of vaccines against infectious bronchitis virus (IBV), bovine coronavirus (BCV), porcine epidemic diarrhoea virus (PEDV), feline infectious peritonitis (FIP), and canine coronavirus (CCV) being used broadly for many decades. As an interesting parallel to SARS-CoV-2 infection, IBV is transmitted via the respiratory route, initially causing an upper respiratory infection in chickens followed by the systemic disease which, depending on the strain, can involve the kidney, reproductive organs, or the intestinal tract. IBV has evolved into an enormous number of variant strains globally and it is important to note that many different serotypes of IBV are present18. These serotypes are sufficiently antigenically distinct that most require unique serotype-specific vaccines to control the disease.

The licensure of IBV vaccines requires not only the demonstration of protection from clinical disease but also a highly significant reduction of virus replication in the trachea. Given its respiratory route of transmission and the requirements to significantly reduce virus present in the respiratory tract, the most effective vaccines are live attenuated viruses, which are delivered by mucosal application, either by spray or in drinking water. Local delivery of live attenuated IBV vaccines induces relatively short-lived local mucosal IgA neutralizing antibody responses in addition to systemic IgA and IgG antibody responses19,20,21. However, the complete mechanism of protection is unclear and is likely to involve cell-mediated immunity specific for other proteins besides the S. In longer-lived birds, responses are boosted by the parenteral delivery of adjuvanted inactivated whole virus vaccines, which extends the duration of immunity significantly and is likely to boost the immune response that has been primed by the mucosally delivered vaccine. Interestingly, the induction of virus-neutralizing serological antibodies against the S protein by parenteral vaccination routes has previously been shown to provide a low level of protection against respiratory IBV challenge22,23,24. It is therefore of particular interest to determine how well the human SARS-CoV-2 vaccine candidates, most of which are designed to be administered parenterally, are able to control upper respiratory tract infection and virus spread, in addition to preventing clinical disease. Initial indications are that these vaccines are successfully reducing human to human spread, though the mechanisms involved require further investigation.

In the controlled experiments in felines, it has been shown that the SARS-CoV-2 virus can readily infect cats and although in most cases no or only a mild disease was detected, the cats can shed the virus for prolonged periods, infecting other cats4. Prevention or limitation of virus replication in infected cats, thereby reducing direct contact transmission in cats, would be useful in preventing the establishment of a reservoir in the feline population and also potentially limit the development or selection of viral mutants in these species. In order to investigate the prevention of transmission between cats, we tested whether a vaccine could protect cats from SARS-CoV-2 challenge and also prevent virus spread between cats under controlled conditions.

For this purpose, we utilized an Alphavirus-based Replicon technology derived from the attenuated TC-83 strain of Venezuelan equine encephalitis virus (VEEV) to express the SARS-CoV-2 S protein.

Several other vaccines based on alphavirus replicons have already been designed such as repRNA-CoV2S25, LUNAR-COV1926, and alphavirus-based DNA-launched self-replicating (DREP)27, all based on the SARS-CoV-2 wild-type spike protein and showing promising antibody responses and T cell activation. Additionally, replicon technology has been tested in numerous species (including humans)28,29 and the VEEV based replicon has been shown to be safe in cats reducing the clinical effects and shed of Feline Calicivirus, which causes an acute respiratory tract infection (Authors’ unpublished observations). Furthermore, good responses have been detected in chickens, dogs, horses, pigs, and cattle with a variety of antigen targets28. The replicon forms the basis of the Sequivity®RNA Particle vaccine platform which is currently licensed in the US for multiple swine applications. In this system, the foreign gene of interest, in this case, SARS-CoV-2 S, is inserted in place of VEEV structural genes generating a self-amplifying RNA capable of expressing the gene of interest upon introduction into cells. The self-amplifying replicon RNA directs the translation of large amounts of protein in transfected cells, reaching levels as high as 15–20% of total cell protein30. As the replicon RNA does not contain any of the VEEV structural genes, the RNA is propagation-defective. The replicon RNA can be packaged into replicon particles (RP) by supplying the VEEV structural genes in trans in the form of promoter-less capsid and glycoprotein helper RNAs and, when the helper and replicon RNAs are combined and co-transfected into cells, the replicon RNA is efficiently packaged into single-cycle, propagation-defective RP which are used in the vaccine formulation31. RP vaccines have been shown to induce both innate and adaptive immune responses including virus neutralizing antibodies and T cell responses28. Of significant importance is the fact that this system can be employed very rapidly with materials sufficiently available for deployment of vaccine within weeks.

The structural conformation and cellular localization of the SARS-CoV-2 S protein have been found to be important for the induction of a protective immune response32,33,34. We therefore generated and tested, alongside the wild type variant, five S antigens harboring several types of mutations designed to stabilize the metastable S protein in its prefusion conformation and to increase its cell surface localization. These S antigens were evaluated for protein expression studies in vitro, and were used to generate VEEV RP-based vaccines for testing their immunogenicity in guinea pigs. We further focused on two of the antigen candidates producing either the wild type S antigen (Spike Wt) or an optimized S antigen (Spike Opt), containing the majority of the stabilizing mutations. We compared both antigens—delivered via a plasmid DNA-launched replicon RNA (DREP) vector vaccine or the RP-based vaccine—for their efficacy in eliciting humoral and cellular immune responses. The Spike Opt RP vaccine was selected and evaluated in a cat vaccination-challenge experiment for its ability to protect against SARS-CoV-2 infection and/or prevent transmission to in-contact non-vaccinated cats.

Results

SARS-CoV-2 Spike antigen design

The recent SARS-CoV-2 vaccine efforts have shown that stabilizing the pre-fusion form of the S protein enhances immunogenicity of the antigen in the mRNA and vector-based vaccines33,35. Therefore, we designed five SARS-CoV-2 S antigen forms with combinations of mutations that potentially enhance its stability or cell surface expression, including inactivation of the furin cleavage site (FCSmut), the double-proline stabilizing mutations (2P), deletion of the C-terminal domain (∆CTD) and/or the replacement of the CTD and the transmembrane domain (TM) with the counterparts of the G protein of the vesicular stomatitis virus (VSV) (Fig. 1).

Fig. 1: Schematic representation of the SARS-CoV-2 S antigen designs.
figure1

The position of the wild-type signal peptide (SP), the S1 and S2 subunit, the furin cleavage site (FCS), the transmembrane (TM), and cytoplasmic tail domain (CTD) are indicated. Constructs contain various alterations including FCS mutations (FCSmut: R682A/R683A), proline stabilizing mutations (2P: K986P/V987P), deletion of the CTD, and replacement of the TM/CTD by that of the vesicular stomatitis virus G protein (VSV).

In vitro analysis of modified Spike antigens and immunogenicity study in guinea pigs

The surface expression of all six S variants generated from pCAGGS2 plasmids on transfected cells was confirmed by immunofluorescence assay (Fig. 2A) and quantified using flow cytometry (Fig. 2B). A trend towards higher cell surface expression was observed for S forms that included the TM/CTD of VSV-G, especially in combination with the stabilizing 2P mutations (Spike FCSmut-VSV and Spike FCSmut-VSV).

Fig. 2: In vitro expression of SARS-CoV-2 S variants and humoral immune responses in vaccinated guinea pigs.

A Antigen expression levels and localization on the surface of HeLa cells using immunofluorescence microscopy (scale bar 50 µm). Nuclei were stained with Hoechst (blue colour) and S proteins with an anti-RBD targeting antibody (green colour). B Quantification of antigen expression levels on the cell surface of plasmid transfected HEK293T cells using FACS by measuring the mean fluorescence intensity (MFI) at 24 h post-transfection. Immunostaining for the spike protein was the same as in (A). C SARS-CoV-2 S antibody levels in sera of VEEV RP vaccinated guinea pigs, collected at day 56 after vaccination/boost, was measured by ELISA using SARS-CoV-2 S RBD (solid colour) or S ectodomain (pattern) as capturing antigens. Shown are the half-maximal effective concentrations (EC50) values of sera collected at the end of the study (expressed as fold dilution). D Neutralizing antibody titers against SARS-CoV-2 S pseudotyped VSV determined on Vero E6 cells, and expressed as the half-maximal inhibitory concentrations (IC50, fold serum dilution). E SARS-CoV-2 surrogate virus neutralization test measuring antibody-mediated blockage of S-ACE2 receptor interaction using 10.000-fold diluted serum samples of guinea pigs, collected at the end of the study.

To assess the immunogenicity of the S variants, we immunized guinea pigs using vaccines based on the VEEV replicon particle (RP) system. Animals were immunized with 1 × 107 VEEV RPs via the intramuscular route at day 0, 21, and 42 of the study, and serum for analysis was collected at day 56. Compared to the wild-type S antigen, inactivation of the FCS (the other five S variants with FCSmut) leads to a dramatic increase in ELISA antibody titers against the receptor-binding domain (RBD) or the S ectodomain (SED) (Fig. 2C). Additional mutations introduced into the S protein did not further increase immunogenicity significantly. Neutralizing antibody titers - measured by a commercial SARS-CoV-2 surrogate virus neutralization assay or a pseudovirus neutralization assay—corroborated the ELISA results (Figs. 2D, E). Relative to wild type-S antigen, a trend towards higher neutralization titers were observed for sera of animals immunized with the VEEV RP vaccine producing the S antigens containing a mutated FCS, replacement of the TM/CTD for that of VSV-G in combination with the 2P stabilizing mutations (Spike FCSmut-2P-VSV). Based on the cell surface and immunogenicity data we selected this SARS-CoV-2 S form as the lead vaccine antigen, and it will be further referred to as Spike Opt.

Immunogenicity study of VEEV DREP and VEEV RP vaccine candidates in guinea pigs

Immunogenicity of the Spike Wt and Spike Opt antigens was assessed in a guinea pig model using a plasmid DNA-launched replicon RNA (DREP) based vector vaccine or the VEEV RP vector vaccine, following intramuscularly administration (Fig. 3A). After prime-boost vaccination, all animals showed seroconversion as assessed by both the RBD as well as S ectodomain (SED) indirect ELISA. The DREP vaccine immunizations led to inferior titers as compared to the VEEV RP vaccines (Fig. 3B, C). In line with the previous guinea pig study, the wild-type S induced lower antibody titers compared to the Spike Opt antigen.

Fig. 3: Immunogenicity of vaccine candidates in a guinea pig model.
figure3

A Timeline of animal handlings. V = vaccination with plasmid DNA-launched replicon RNA (DREP) or VEEV replicon particles (RP), B = blood sampling. B, C Indirect ELISA performed using SARS-CoV-2 S RBD (left) or S ectodomain (right) as capturing antigens. Shown are the EC50 values of sera (expressed as fold dilution) from guinea pigs exposed to the wildtype S antigen (Spike Wt) or the optimized S antigen (Spike Opt). D SARS-CoV-2 surrogate virus neutralization (VN) assay performed using 1.000-fold diluted serum samples collected after prime-boost vaccination at day 34 and expressed as percent inhibition. E Results of lymphocyte stimulation test (LST) using blood collected on day 70/71. Purified SARS-CoV-2 S1 antigen was used to stimulate isolated lymphocytes and proliferation was measured 96 h post-stimulation. Gating-procedure in Supplementary Fig. 1.

Neutralizing antibody titers in sera of immunized animals were quantified using the SARS-CoV-2 surrogate virus neutralization (VN) test. Clearly higher surrogate VN titers were induced by the Spike Opt antigen compared to the Spike Wt antigen in both vaccine platforms (Fig. 3D). The VEEV RP vector platform is known for its efficient induction of both humoral, as well as cellular responses28. To assess the level of cellular responses induced by the vaccine candidates delivered as VEEV RPs or DREP, a third immunization was performed and seven days later lymphocytes were isolated for a lymphocyte stimulation test (LST). In contrast to the differences in humoral responses between the two vaccine platforms, DREP immunizations lead to comparable T-cell activation upon stimulation with high concentration (5.0 µg/ml) of SARS-CoV-2 S1 antigen compared to the VEEV RPs (Fig. 3E). Interestingly, at low concentration of SARS-CoV-2 S1 antigen (0.16 µg/ml) the level of reactive T-cells seems to be higher in DREP vaccinated animals compared to VEEV RP vaccinated animals. Concerning the Spike Wt and Spike Opt antigens, a slight increase was observed in levels of SARS-CoV-2 S1 specific T-cell differentiation for the Spike Opt antigens when stimulated with low concentration SARS-CoV-2 S1 antigen, but this difference is no longer noticeable when stimulations were performed with 5 µg of S1 (bars with yellow stripes).

To determine whether the humoral immune response also resulted in mucosal immunity, tracheal swabs were taken at the end of the experiment. Interestingly, surrogate VN titers were detected in the trachea swabs, and the levels correlated with the systemic antibody levels with superior titers for the Spike Opt antigen compared to the Spike Wt antigen (Fig. 3F). These antibody titers suggest that parenteral vaccination could induce protective mucosal immunity.

Cat vaccination-challenge study

To determine vaccine efficacy, cats were either vaccinated with a VEEV RP vaccine producing EGFP (Control), the optimized SARS-CoV-2 S antigen (Spike Opt) or remained non-vaccinated (sentinels). Three weeks post booster vaccination, cats were exposed to a mucosal SARS-CoV-2 challenge using the intranasal and oral routes, and samples were taken as outlined in Fig. 4A.

Fig. 4: Vaccination-challenge experiment in cats.
figure4

A Timeline of animal handlings. V = vaccination, B = blood sampling, O = oropharyngeal swabs, N = nasal swab, (all) = all animals, (ch) = only challenged animals, (sen) = only sentinel animals. B Serum neutralizing antibody titers determined using a SARS-CoV-2 virus neutralization (VN) assay at 21- and 42-days post vaccination (d.p.v.) in the non-vaccinated (Control) or Spike Opt vaccinated cats. CSerum neutralizing antibody titers determined using a VN test using the SARS-CoV-2 B.1.1.7 variant virus at 42-days post vaccination in the non-vaccinated (Control) or Spike Opt vaccinated cats. DSerum neutralizing antibody titers were determined using a SARS-CoV-2 virus neutralization assay at the day of challenge, 42-days post vaccination and 14 days post challenge and shown for challenged control vaccinated animals (solid grey), non-vaccinated sentinel animal co-housed with control vaccinated animals (grey with pattern), Spike Opt antigen vaccinated animals (solid orange), or non-vaccinated sentinel animals co-housed with Spike Opt antigen vaccinated animals (orange with pattern). SARS-CoV-2 virus titers were measured in oropharyngeal swabs (E) or nasal swabs (F) at 1−8 days post challenge (d.p.c.) expressed in PFU/ml. All experiments were performed on Vero E6 cells.

Following vaccination, no adverse reactions were detected in any of the cats at any time point. The VEEV RP vaccine producing the Spike Opt antigen was able to induce virus-neutralizing antibody titers in all cats after a single vaccination, which was boosted after the second vaccination and maintained levels until the challenge 3.5 weeks later (Fig. 4B). The vaccine-induced antibodies were also potently neutralizing the SARS-CoV-2 B.1.1.7 variant (Fig. 4C), a strain that recently has been associated with clinical manifestation in cats14. Control and non-vaccinated sentinel animals remained seronegative at all times up until the challenge. Both challenged and sentinel cats did not demonstrate any clinical signs post challenge. However, nine out of ten control challenged cats shed virus orally (Fig. 4E) and nasally (Fig. 4F) one day after the challenge and for at least 3 days during the observation period. These data show that the mucosal SARS-CoV-2 challenge results in efficient virus replication in the respiratory tract. Higher and more consistent virus shed was detected from the nasal washes whereas the oropharyngeal swabs demonstrated a less consistent pattern, the reason for this being unknown. Interestingly, virus shed was also detected from the nasal washes in two of the non-vaccinated sentinels placed with the control animals one day after the challenge. Moreover, all five sentinel animals housed with the challenged control cats shed virus via the oral route for at least two days demonstrating the efficient spread of the virus from control challenged to sentinel animals (Fig. 4E).

None of the vaccinated cats shed any detectable virus orally (Fig. 4E) or nasally (Fig. 4F) at any time point after the challenge. The results suggest that the vaccine may have prevented infection. Also, no virus was detected in the non-vaccinated sentinels housed with the vaccinated cats, as would be expected considering the lack of challenge virus replication in the vaccinated cats. Analysis of virus-neutralizing antibody titers post-challenge confirmed the findings that both control challenged and sentinel animals were efficiently infected (Fig. 4D). In contrast, no seroconversion was observed in the sentinel animals housed with the vaccinated cats. Thus, the VEEV RP vaccine producing the Spike Opt antigen appears to induce protective immunity and prevent transmission from infected to naïve cats.

Discussion

With the ongoing pandemic and reports of naturally occurring SARS-CoV-2 infections of a variety of animal species, it is important to understand the epidemiology of this virus in these animal populations especially with regards to the establishment of potential reservoirs, mutations, and transmission within and to other species. SARS-CoV-2 infections in humans can be transmitted to cats and it has been hypothesized that cat-to-cat transmission of virus can take place in a natural setting1. It was previously demonstrated that SARS-CoV could infect and spread between cats23, but the complete epidemiological picture of feline infection was not fully understood with the rapid eradication of SARS-CoV from humans before it reached a pandemic situation. The situation with SARS-CoV-2 is different as it has become a global issue with the likelihood of becoming endemic in the human population. Whilst an infected cat is considered a low risk for SARS-CoV-2 transmission to humans, to other cats and other species, considering that infected cats shed virus for prolonged periods which can potentially be aerosolised gives credence to the possibility that cats may play some role in the viral epidemiology either by transmitting the virus onwards, enabling further mutations or acting as a virus reservoir. Although routine vaccination of cats is not proposed, should the epidemiological situation change, the availability of a vaccine that can be rapidly produced, updated, and which reduces or prevents viral replication and transmission between cats and other animals will be useful. Furthermore, a vaccine that could be used in a range of susceptible animal species would be preferable.

We developed a safe and effective VEEV replicon particle-based SARS-CoV-2 vaccine for cats. Our data demonstrate that prime-booster immunization with replicon particles encoding a prefusion stabilized S immunogen induced neutralizing antibody responses in sera and mucosal tissues, and provided full protection against SARS-CoV-2 challenge in all cats.

Based on the immunization studies, the S antigen design in combination with the furin cleavage site mutation (FCSmut) appeared most critical to the robust induction of a neutralizing antibody response in both guinea pigs and cats, indicating that this prefusion S stabilizing modification can increase immunogenicity. In the guinea pig experiments, it was interesting to note that intramuscular vaccination induced some level of mucosal antibody titers, which was somewhat surprising and is likely to be a wash over from serological induction. It remains to be established whether these antibodies might contribute to the protective immunity that has been observed for this vaccine candidate in cats.

The optimized S RP vaccine successfully induced a virus-neutralizing antibody response in all vaccinated cats after a single vaccination which was boosted upon a second vaccination. Furthermore, the vaccine was able to prevent infection in all vaccinated cats as demonstrated by the lack of virus re-isolation post-challenge. Although there was a strong induction of a serological response in the cats it was not investigated, as was demonstrated in the guinea pig experiments, whether neutralizing antibody was present in the respiratory tract. Furthermore, we did not examine the role of cell-mediated immunity in the prevention of infection of cats nor were we able to extend the experiments to investigate the duration of the immune response induced. The ability to induce local protection from parenterally administered coronavirus vaccines is not well established and in certain veterinary respiratory coronavirus infections mucosally-applied live attenuated vaccines are used to reduce the viral replication at the site of initial infection. These live vaccines induce a relatively brief period of protection, so they are boosted by inactivated adjuvanted whole virus vaccines to establish longer immunity. The use of inactivated vaccines alone in these veterinary settings is not as effective at protecting the local respiratory tract as the live priming inactivated boost approach24. For this reason, it is reassuring that a parenterally administered vaccine did appear to provide respiratory protection in the feline model. Future work would be needed to establish whether a single vaccine dose would also be sufficient to protect the cats from infection. Furthermore, the optimal inoculation schedule has not yet been established for this vaccine nor has the duration of immunity that can be induced and whether the ability to prevent infection and spread persists over this time.

This work demonstrates the utility of the VEEV strain TC-83-based replicon particle vaccine platform (Sequivity®). RP vaccines based on VEEV have previously been shown to protect cats against viral diseases including some respiratory protection against clinical signs and virus shed in a feline calicivirus infection model, and in addition have been shown to be effective in multiple species including dogs, horses, pigs, cattle, chickens and ducks28, authors’ unpublished observations. Furthermore, VEEV based RP vaccines expressing the S proteins from other coronaviruses have been shown to induce virus-neutralizing antibodies36. The advantages of RP-based technology is that vaccines can be rapidly prepared if the gene of interest is known to encode a protective antigen. This vaccine platform is safe-by-design as the RP vaccines undergo a non-productive cycle of replication in which replicon RNA but no virus is replicated and no adjuvants are required31.

Thus far Rhesus macaques, hamsters, and ferrets have been utilized as natural animal models for SARS-CoV-2. In these animals, infection is usually asymptomatic or induces mild clinical disease. As SARS-CoV-2 also induces asymptomatic infections in cats, this species may also provide a means to study virus transmission, especially via aerosols and vaccine design aimed at preventing initial infection in the respiratory tract. In some respects, infection of cats may mimic the majority of human infections which are asymptomatic.

This work demonstrates that a VEEV replicon-based vaccine expressing the stabilized SARS-CoV-2 S protein was able to induce high levels of virus-neutralizing antibodies in serum of vaccinated cats and that the induced response was able to prevent infection of the upper respiratory tract, thereby preventing onward transmission to other cats.


Article classification: Biological abstract
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