key: cord-0878528-2kzqfufi
authors: Kurup, Drishya; Malherbe, Delphine C.; Wirblich, Christoph; Lambert, Rachael; Ronk, Adam J.; Diba, Leila Zabihi; Bukreyev, Alexander; Schnell, Matthias J.
title: Inactivated rabies virus vectored SARS-CoV-2 vaccine prevents disease in a Syrian hamster model
date: 2021-01-20
journal: bioRxiv
DOI: 10.1101/2021.01.19.427373
sha: 4a34f4c09888cb271dab5f885c728f4b5e6736e9
doc_id: 878528
cord_uid: 2kzqfufi

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus that has caused a worldwide pandemic. Although human disease is often asymptomatic, some develop severe illnesses such as pneumonia, respiratory failure, and death. There is an urgent need for a vaccine to prevent its rapid spread as asymptomatic infections accounting for up to 40% of transmission events. Here we further evaluated an inactivated rabies vectored SARS-CoV-2 S1 vaccine CORAVAX in a Syrian hamster model. CORAVAX adjuvanted with MPLA-AddaVax, a TRL4 agonist, induced high levels of neutralizing antibodies and generated a strong Th1-biased immune response. Vaccinated hamsters were protected from weight loss and viral replication in the lungs and nasal turbinates three days after challenge with SARS-CoV-2. CORAVAX also prevented lung disease, as indicated by the significant reduction in lung pathology. This study highlights CORAVAX as a safe, immunogenic, and efficacious vaccine that warrants further assessment in human trials.

More than 150 vaccines against SARS-CoV-2 are in preclinical trials, and over 51 candidate vaccines are in human trials (1). They include adenovirus and other viral vector-based vaccines and protein-, DNA-, and mRNA-based vaccines (2) .

These vaccine approaches have different advantages and disadvantages. mRNA vaccines, including the 2 U.S. FDA-approved coronavirus vaccines from Moderna and Pfizer-BioNTech, can be produced efficiently, but they can be costly to produce and have temperature sensitivity (3) ; furthermore, questions remain unanswered about the longevity of the immune response with mRNA vaccines and if they block transmission. Meanwhile, DNA-based vaccines benefit from temperature stability and low production costs, but their immunogenicity has been a concern, and the need for multiple vaccinations challenges their feasibility (4). Virus-like particle (VLP)-and recombinant protein-based vaccines have an excellent safety advantage because they do not replicate in the host, and often they can also be made more temperature stable.

However, VLP-based vaccines are not always as immunogenic as replication-competent or replication-deficient viral vector vaccines and often require an adjuvant to increase their immunogenicity to an adequately protective level (5, 6) . Finally, viral vector-based vaccines the advantage of being typically cheaper to produce, efficacious after a single vaccination, and often highly immunogenic (1, 7-9), but those based on live viral vaccines sometimes fail because of safety concerns and production scalability, and they usually require a low storage temperature (8) . The disadvantages of viral vector vaccines are largely overcome, however, when they are based on an inactivated virus.

Using this approach, we have developed an inactivated viral vector vaccine against SARS-CoV-2 that is based on the rabies virus (RABV). We have previously utilized this approach to develop inactivated RABV-based vaccines for several other human pathogens (e.g., Ebola virus, Marburg virus, Lassa Fever virus and others) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) . These rabies virus-based vaccines have been proven to be highly immunogenic and protective against several emerging viral infections and bacterial toxins, as well as safe and temperature stable (28) . The RABV vaccine itself has been safely used for decades in more than 100 million people, including children, pregnant women, and the elderly, and proven to result in long-lasting immunity (29) . CORAVAX, our rabies vectored SARS COV-2 vaccine, expresses the S1 domain of the SARS-CoV-2 spike (S) protein fused to part of the N terminal domain of the RABV glycoprotein (G) and is incorporated in RABV particles. Mice immunized with adjuvanted CORAVAX developed potent neutralizing antibodies about 5-10 times higher than the virus-neutralizing antibodies (VNA) detected in convalescent sera from SARS-CoV-2 infected people (30) . Here, to learn more, we evaluated the efficacy of CORAVAX in the Syrian hamster model for SARS-CoV-2, which is currently considered the best model to study COVID-19 therapies and therapeutics.

Our results showed that two immunizations with the adjuvanted CORAVAX vaccine induced high VNA and cleared infectious SARS-CoV-2 from the lung and the nasal turbinates on day 3 post challenge. Moreover, the pathology induced by SARS-CoV-2 was reduced with only some early infiltration of cells into the lung tissue. Taken together, this study indicates that CORAVAX warrants further development as a disease-preventing human vaccine.

CORAVAX a rabies vectored SARS COV-2 vaccine was generated by inserting the S1 subunit of the SARS-CoV-2 Spike into the SAD-B19 rabies vaccine strain. To promote the incorporation of the S1 domain, we prepared a fusion protein between SARS-CoV-2 S1 and RABV G. Toward this approach, the N-terminal 682 aa of SARS-CoV-2 S1 were fused to a truncated RABV glycoprotein, which comprises 31 aa of the ectodomain (ED) of RABV G and the complete cytoplasmic domain (CD) and transmembrane domain of RABV G to allow chimeric glycoprotein incorporation into RABV virions. We previously showed that CORAVAX induces high virus neutralizing antibodies as a live or inactivated vaccine in mice (30) . Here we extend these studies in the more relevant SARS CoV-2 Syrian Hamster model of severe disease.

To evaluate the immunogenicity and efficacy of the CORAVAX vaccine in golden Syrian hamsters, we vaccinated each group comprising 12 animals with either 10 g of inactivated CORAVAX or control vaccine FILORAB1 (rabies vectored Ebola vaccine) adjuvanted with MPLA-AddaVax in a 100 L injection volume via the intramuscular route ( Figure 1A) . The animals received a prime on day 0 and a booster on day 28. Blood was collected on days -2, 26, and 56 ( Figure 1B ). On day 60, vaccinated and control hamsters were challenged intranasally with a dose of 10 5 PFU of the SARS-CoV-2 isolate USA_WA1/2020 (31) .

In the next step, sera from immunized hamsters and controls were assayed for SARS CoV-2 specific antibody responses by ELISA specific for SARS-CoV-2 S1 and receptor binding site (RBD) (Figure 2 ). High titers of SARS-COV-2 S1 specific IgG responses were detected in the CORAVAX vaccinated hamsters on day 26 with no significant differences in antibody titers on day 56 ( Figure 2A ). As expected, no SARS-CoV-2 S1 immune response was detected in the control animals vaccinated with the EBOV vaccine FILORAB1. As previously seen for other inactivated and adjuvanted RABV-based vaccines, CORAVAX induced a Th1 biased immune response as indicated by the high SARS CoV-2 S1 IgG2/3 responses detected on days 26 and 56 ( Figure 2B ). We could not detect S1 specific IgG1 responses in any of the hamsters (data not shown). Similar to the responses detected by ELISA to SARS CoV-2 S1, we observed high titers of SARS-CoV-2 VNA with mean titers of 976  SD 685 on day 56 ( Figure 2C ). Of note, no VNA against SARS-CoV-2 were detected in FILORAB1 immunized animals before the challenge ( Figure 2C ).

We next analyzed the immune responses post-challenge (p.c.). By day 63, 3 days p.c., we observed a significant reduction (P=0.0069) in S1 IgG responses in CORAVAX vaccinated animals, while no S1-specific IgG responses were observed in the controls hamsters (FILORAB1) (Figure 2A ,C). By day 75, both in CORAVAX and FILORAB1 vaccinated and challenged hamsters, S1-specific IgG and VNA were detected (Figure 2A ,B,C). However, both S1-specific IgG and VNA were significantly higher in the CORAVAX vaccinated animals on day 75 (Figure 2A, C) . Moreover, p.c. CORAVAX vaccinated animals indicated a more robust Th1biased immune response compared to FILORAB1 control animals, as indicated by the IgG2/3 responses ( Figure 2B ). In addition to the antibodies directed against the receptor-binding domain (RBD) SARS-CoV-2, high RBD-specific IgG titers were detected in the CORAVAX vaccinated animals on day 56 as well as p.c. on days 63 and 75. Interestingly, the RBD IgG titers were undetectable in the FILORAB1 vaccinated controls on day 63 but were higher than the ELISA titers detected in CORAVAX vaccinated animals on day 75, challenging the dogma that high ELISA titer against RBD predict the VNA against SARS-CoV-2 ( Figure 2D ).

We also analyzed the VNA induced by the two vaccines against RABV. CORAVAX is a vaccine against both SARS-CoV-2 and RABV, and in large part of the world RABV is still a significant problem, <<annually?>> killing about 55,000 people, mostly children. We detected a titer of anti-RABV neutralizing antibodies above the WHO's 0.5 IU standard in both the FILORAB1 and CORAVAX vaccinated animals, with no significant differences ( Figure 3 ).

The hamsters were challenged intranasally on day 60 with 10 5 PFU of SARS-CoV-2 isolate USA_WA1/2020 and were monitored for up to 15 days. The CORAVAX vaccinated animals showed significantly less weight loss than the FILORAB1 controls (P=0.0098), which lost more than 10% weight and recovered only at day 11 ( Figure 4 ). At days 3 and 15 p.c., half of the hamsters in each study group were euthanized, and lungs and nasal turbinates were harvested and virus isolated to determine viral loads by plaque reduction assay ( Figure 5A, B) .

Additionally, the number of viral copies was analyzed by RT-qPCR assay ( Figure 5C , D). The lack of weight loss coincided with the absence of any infectious virus in the lungs and nasal turbinates of the CORAVAX vaccinated animals on days 3 and 15 p.c. (Figure 5A and B).

In contrast to the CORAVAX immunized animals, the FILORAB1 controls had high titers of infectious virus in the lungs and nasal turbinates on day 3 p.c.. As expected, both groups cleared the SARS-CoV-2 15 days.

A similar trend between the two groups was detected when RNA copies of SARS-CoV-2 were analyzed via RT-PCR. CORAVAX vaccinated animals had significantly reduced RNA copies in the lungs and nasal turbinates at necropsy (day 3 and 15 p.c.) than the control FILORAB1 group ( Figure 5C, D) . The presence of viral RNA in the absence of infectious SARS-CoV-2 is wellestablished and based on the stability of the genome of SARS-CoV-2 (32, 33) . However, it should be noted that the ~1000-fold lower copy number in CORAVAX vaccinated animals indicates a significant reduction of viral replication by CORAVAX.

Lung sections were collected from control and vaccinated animals at days 3 and 15 p.c. ( Figure   6 , 7). Sections were scored in a blinded manner. Histopathological changes consistent with viral interstitial pneumonia were noted in all animals, regardless of treatment or time of collection ( Figure 6 , representative pathology pictures; Figure 7A , B mean overall pathology scores).

These included consolidation, widespread alveolar septal thickening, and airway pathology consisting of airway epithelial hyperplasia and accumulation of inflammatory cells in airways, occasionally leading to obstruction of the lumen. On day 3 p.c., CORAVAX vaccinated animals had significantly lower average pathology scores. Specifically, component scores for inflammatory foci size and number and airway pathology were improved ( Figure 7A ). Animals cleared the virus by day 15 ( Figure 5A , B), and consistent with expected tissue damage repair following clearance, we observed reduced pathology in both CORAVAX and the control FILORAB1 vaccinated animals ( Figure 7B ).

There is an urgent need for a safe and effective vaccine against SARS-COV-2 that can be administered to pregnant women, children, elderly and the immunocompromised. The two FDA emergency use authorization (EUA) COVID-19 vaccines, BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna), are based on the mRNA platform have shown promising results with efficacy above 90% (34, 35) . Neither of these EUA COVID-19 vaccines are approved for use in pregnant women, breastfeeding mothers, children below the age of 18, and the immunocompromised (36), and they have yet to demonstrate long-lasting immune responses.

Therefore, other vaccine approaches are still needed.

This study is the first to show the efficacy of a RABV vectored COVID-19 vaccine, CORAVAX, in the Syrian hamster model of severe disease. The rabies vaccine vector has several advantages: 1) it has an excellent safety profile, as it is used as an inactivated vaccine; 2) there is historical evidence of long-term immunity; 3) it can be administered safely and effectively to the vulnerable populations of children, pregnant women, elderly, and immunocompromised; 4) pre-existing rabies immunity does not affect the boosting potential of the vaccine; 5) RABV virions incorporate foreign antigens easily; and 6) RABV-based vaccines show excellent temperature stability (2, 19, 28) .

Our vaccine approaches used only part of the SARS-CoV-2 spike protein. Using S1 rather than the full spike protein as the immunogen ensures the immune responses against the important neutralizing epitopes identified in human convalescent patients recognizing the RBD, NTD S1, and quaternary epitope that bridges the two RBDs (37) . Our use of the S1 in CORAVAX might explain the higher VNA titers in the Syrian hamsters compared to other Covid-19 vaccines utilizing the full spike as the antigen in yellow fever and Ad26 vectors as well as in a DNA vaccine platform (38) (39) (40) .

Several candidate vaccines against Covid-19 utilizing DNA, RNA, viral vectors, inactivated vaccines and subunit vaccine platforms are in various stages of pre-clinical or clinical development (41) . While the immune correlates of protection are yet to be determined, most candidate vaccines trials compare their antibody and neutralizing response to SARS-CoV-2 convalescent sera. Clinical trials with convalescent plasma treatment have shown little or no significant difference in outcomes among SARS-CoV-2 patients compared to placebo group (42, 43) . Also, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity (44) .

CORAVAX induced high SARS CoV-2 S1 and RBD specific antibody and neutralizing titers on day 26 post the prime vaccination. The antibody and neutralizing titers were slightly (but not significantly) increased on day 56 after the boost (on day 28). CORAVAX also induced a strong Th1 biased immune response indicated by the IgG2/3 response before the challenge. Postchallenge, CORAVAX vaccinated animals induced significantly higher S1 IgG titers than the controls that correlated well with the neutralizing antibody response. Interestingly the RBD IgG titers were higher in the control animals at day 15 p.c. than CORAVAX vaccinating animals. The correlation of the S1 IgG titers with that of neutralizing antibody responses in the CORAVAX vaccinated animals suggests that the S1 IgG titers are a better predictor of protection than the RBD IgG titers. Our result aligns with the Chi et al. study that identified non-RBD binding, SARS CoV-2 S1 N terminal domain (NTD) binding neutralizing antibodies isolated from convalescent patients (45) . It has also been suggested that the non-RBD binding S1 antibodies could be restraining the conformational changes of the S protein, thereby preventing viral entry.

Conversely, vaccines that induce only RBD antibodies alone might induce resistance mutations in the virus (46) . CORAVAX induces antibodies against the RBD as well other epitopes of S1.

Antibodies induced in the CORAVAX vaccinated hamsters protected them from weight loss post challenge, while FILORAB1 vaccinated animals showed weight loss. The absence of infectious virus in the lungs and nasal turbinates of the CORAVAX vaccinated animals at day 3 p.c.

suggests that CORAVAX can control viral transmission. Additionally, CORAVAX vaccinated animals showed significantly reduced lung pathology and inflammatory foci in comparison to the control group, suggesting that CORAVAX can dramatically reduce disease in vaccinated animals.

Regarding the adjuvant, we previously showed that GLA-SE adjuvanted FILORAB1 rabies virus-based EBOLA vaccine, protected 100% of NHPs challenged with Ebola, an improvement from the unadjuvanted FILORAB1 vaccine, which was less protective. This protection was attributed to the strong Th1 biased immune response induced by GLA-SE, which is a synthetic TRL4 agonist (20) . To induce a stronger Th1 biased response in our study, we utilized another TRL4 agonist, MPLA-AddaVax, because MPLA is a TLR4 agonist and AddaVax is squalene-based oil-in-water nano-emulsion (similar to SE component in GLA-SE). MPLA has been shown to enhance the immunogenicity and protection of the rabies vaccine with induction of plasma cell responses. MPLA vaccinated mice accelerated the activation of dendritic cells, improving Tdependent B cell responses driving antibody production that skewed towards a strong Th1 bias (47) . As seen for mild COVID-19 patients, a Th1 biased immune response is beneficial for protection protection against disease (48) . Our previous work demonstrated that CORAVAX induced a strong Th1 biased immune response in Balb/C mice by generating higher IgG2a antibodies (30) . In Syrian hamsters, CORAVAX vaccinated animals induced a stronger S1 IgG2/3 response before challenge. More importantly, the CORAVAX vaccinated hamsters mounted a significantly higher S1 IgG2/3 on day 15 p.c. than the FILORAB1 vaccinated animals. We could not detect S1 IgG1 responses, but we could detect RABV-G IgG1 responses, suggesting that S1 IgG1 antibodies are present at low levels or absent.

In conclusion, our RABV-vectored COVID-19 vaccine CORAVAX is efficacious and is able to prevent viral replication and reduce disease in Syrian hamsters. CORAVAX also serves as a vaccine against RABV because it induces high RABV VNA. Future studies assessing the efficacy of a single CORAVAX vaccine will be performed since we did not see a dramatic booster effect in immune responses among the hamsters. CORAVAX production should be relatively easy as it would follow the existing RABV vaccine manufacturing facilities and technologies. Additionally, the use of the adjuvant might allow for dose sparing. These results warrant further examination of CORAVAX in clinical trials to be conducted by Bharat Biotech International Ltd.

The following SARS-CoV-2 specific human monoclonal antibodies were kindly provided by 

Recombinant RABV were recovered, purified, inactivated, and titered. Briefly, X-tremeGENE 9 transfection reagent (Millipore Sigma, Cat# 6365809001) was used to cotransfect the full-length viral cDNA clone encoding CORAVAX along with the plasmids encoding RABV N, P, and L proteins and the T7 RNA polymerase into BEAS-2B human lung cells in 6-well plates (RABV). 

The studies were carried out in a strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Research Council. UTMB is an AAALAC-accredited institution and all animal work was approved by the IACUC Committee of UTMB. All efforts were made to minimize animal suffering and all procedures involving potential pain were performed with the appropriate anesthetic or analgesic. The number of hamsters used was scientifically justified based on statistical analyses of virological and immunological outcomes.

Seven-week-old golden Syrian female hamsters (Envigo) were anesthetized with 5% isoflurane prior to immunization and blood collections and with ketamine/xylazine prior to the SARS-CoV-2 challenge. On day 0, 12 animals per group were inoculated with 10 g of CORAVAX or On day 60, vaccinated and control animals were exposed intranasally to the targeted dose of 

To determine antibody responses to the S protein of SARS-CoV-2, an indirect ELISA was developed utilizing purified S1 or receptor binding domain (RBD) protein. The production of the recombinant proteins is described above. Humoral responses to SARS-CoV-2 S1 and RBD protein were measured by an indirect ELISA. We tested individual hamster sera by enzymelinked immunosorbent assay (ELISA) for the presence of IgG specific to SARS-Cov-2 S1 or RBD. In order to test for anti-SARS CoV-2 S1 humoral responses, we produced soluble S1 or RBD as described above. The two recombinant proteins were resuspended in coating buffer (50 mM Na 2 CO 3 [pH 9.6]) at a concentration of 0.5 μg/mL of S1 or 2 μg/mL of RBD, and then they were plated in 96-well ELISA MaxiSorp plates (Nunc) at 100 μl in each well. After overnight incubation at 4 °C, plates were washed 3 times with 1× PBST (0.05% Tween 20 in 1× PBS), followed by the addition of 250 μl blocking buffer (5% dry milk powder in 1× PBST) and incubation at room temperature for 1. 

Sera collected from animals were tested for neutralizing capabilities against SARS-CoV-2.

Briefly, serum samples were heat-inactivated (30 min at 56°C), and then 10-fold diluted sera were further diluted in a 2-fold serial fashion, and 60 µl of each serum dilution was mixed with 60 µl of SARS-CoV-2-mNG (200 PFU) (49) . The serum/virus mixtures were incubated for 1 h at 37°C. 100 µl of the serum/virus mixtures were then transferred to Vero E6 cell monolayers in flat-bottom 96-well plates and incubated for 2 days at 37°C. Virus fluorescence was measured with a Cytation Hybrid Multi-Mode reader at 488 nm (Biotek Instruments).

Animals were euthanized on days 3 and 15 p.c., and lungs and nasal turbinates were harvested. Right lungs and nasal turbinates were placed in L15 medium supplemented with 10% fetal bovine serum (Gibco) and Antibiotic-Antimycotic solution (Gibco). Tissues were homogenized using the TissueLyser II system (Qiagen) and tissue homogenates were aliquoted and stored at -80ºC. Tissue homogenates were titrated on Vero E6 cell monolayers in 24-well plates to determine viral loads. Plates were incubated 3 days at 37°C before being fixed with 10% formalin and removed from containment. Plaques were visualized by immunostaining with 1 µg/mL cocktail of 37 SARS-CoV-2 specific human antibodies kindly provided by Distributed Bio. As the secondary antibody, HRP-labeled goat anti-human IgG (SeraCare) was used at dilution 1:500. Primary and secondary antibodies were diluted in 1X DPBS with 5% milk.

Plaques were revealed by AEC substrate (enQuire Bioreagents).

Tissue homogenates were mixed with TRIzol Reagent (Life Technologies) at a 1:5 volume ratio of homogenate to TRIzol. The RNA extraction protocol for biological fluids using TRIzol Reagent was followed until the phase separation step. The remaining RNA extraction was done using the 

Following euthanasia, necropsy was performed, gross lesions were noted, and left lungs were placed in 10% formalin for histopathological assessment. After a 24-h incubation at 4ºC, lungs were transferred to fresh 10% formalin for an additional 48-hour incubation before removal from containment. Tissues were processed by standard histological procedures by the UTMB Anatomic Pathology Core, and 4 μm-thick sections were cut and stained with hematoxylin and eosin. Sections of lungs were examined for the extent of inflammation, type of inflammatory foci, and changes in alveoli/alveolar septa/airways/blood vessels in parallel with sections from uninfected or unvaccinated lungs. The blinded tissue sections were semi-quantitatively scored for pathological lesions using the criteria described in Supplemental 

Statistical analyses were performed with GraphPad Prism for Windows (version 6.07). P<0.05

was considered significant. P > 0.123 (ns), P < 0.033 (*), P < 0.002 (**), P < 0.001 (***).

Work with SARS-CoV-2 was performed in the BSL-4 facilities of the Galveston National Laboratory according to approved standard operating protocols.

This work was supported in part by a grant from Bharat Biotech, India, and the State of Hamsters were vaccinated at day 0 and day 28, challenged intranasally with 10 5 PFU SARS-CoV-2 at day 60. Percent change in body weight. CORAVAX vaccine group is shown in blue and FILORAB1 group in black. N = 12 for FILORAB1 group (6 hamsters euthanized at day 3 p.c.) and N = 11 for CORAVAX group (6 hamsters euthanized at day 3 p.c.). Body weight P value determined by Wilcoxon test. P > 0.123 (ns), P < 0.033 (*), P < 0.002 (**), P < 0.001 (***). 

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