Most people with COVID-19 have relatively mild disease, but a subset of people develop severe pneumonia and respiratory failure, potentially leading to death.
Beth Israel Deaconess Medical Center (BIDMC) immunologist Dan H. Barouch, MD, Ph.D., and colleagues showed in recently published previous work that a candidate COVID-19 vaccine raised neutralizing antibodies that robustly protected non-human primates (NHPs) against SARS-CoV-2, the virus that causes COVID-19.
Now, in new research published to-day in Nature Medicine, Barouch and colleagues demonstrated that the optimal vaccine elicited robust im-mune response in Syrian golden hamsters and prevented severe clinical disease – including weight loss, pneumonia and death.
“We recently reported that an Ad26-based SARS-CoV-2 vaccine provided robust protection in rhesus ma-caques, and this vaccine is currently being evaluated in humans,” said Barouch, who is Director of BIDMC’s Center for Virology and Vaccine Research.
“However, nonhuman primates typically don’t get severe clinical disease, and thus it was important to study whether this vaccine could prevent severe pneumonia and death due to SARS-CoV-2 in hamsters, which are more susceptible to clinical disease.”
The vaccine – developed through a collaboration between BIDMC and Johnson & Johnson (J&J) – uses a common cold virus, called adenovirus serotype 26 (Ad26), to deliver the SARS-CoV-2 spike protein into host cells, where it stimulates the body to raise immune responses against the coronavirus.
Barouch’s group and J&J developed a series of vaccine candidates designed to express different variants of the SARS-CoV-2 spike protein, which is the major target for neutralizing antibodies.
In the current study, the researchers immunized Syrian golden hamsters with a single injection of the Ad26-based SARS-CoV-2 vaccine, which induced neutralizing antibodies in all animals. Four weeks later, the ani-mals were exposed to a high dose of SARS-CoV-2.
Vaccinated animals lost less weight and had less virus in their lungs and other organs than unvaccinated control animals. Vaccinated animals also demonstrated lower mortality. Moreover, the researchers found that neutralizing antibody responses were inversely correlated with weight loss and viral loads in respiratory tissues.
The Ad26.COV2.S vaccine is currently being evaluated in clinical studies to establish the performance of the vaccine candidate in humans.
“This hamster model of severe COVID-19 disease should prove useful to complement current nonhuman pri-mate models in the evaluation of candidate vaccines and therapeutics,” said Barouch, who is also the William Bosworth Castle Professor of Medicine at Harvard Medical School, a member of the Ragon Institute of MGH, MIT, and Harvard, and the co-leader of the vaccine working group of the Massachusetts Consortium on Pathogen Readiness.
In July 2020, investigators at BIDMC and other institutions initiated a first-in-human Phase 1/2 clinical trial of the Ad26.COV2.S vaccine in healthy volunteers. Kathryn E. Stephenson, MD, MPH, is the principal investiga-tor for the trial at BIDMC, which is funded by Janssen Vaccines & Prevention, B.V., a pharmaceutical re-search arm of Johnson & Johnson.
Pending clinical trial outcomes, the Ad26.COV2.S vaccine is on track to start a phase 3 efficacy trial in up to 60,000 participants in September 2020.
Antigen Selection and Engineering
Coronaviruses Encode Multiple Structural and Non-structural Proteins that Could Potentially Serve as Immunogens for a SARS-CoV-2 Vaccine
The best characterized proteins are S, N, M, and E. S has most commonly been utilized in coronavirus vaccine studies, due to its pivotal role in mediating viral entry into cells (Song et al., 2019). Mature S is a trimeric class I fusion protein located on the surface of the virion. Many coronaviruses proteolytically process S into the S1 and S2 domains.
The S1 fragment contains the receptor binding domain (RBD) and the S2 fragment contains the fusion peptide, which are responsible for receptor binding and cell fusion, respectively. For SARS-CoV, S has been demonstrated to be the primary target of neutralizing antibodies.
In mouse models of SARS-CoV, passive transfer and vaccine studies have shown that S-specific antibodies confer protective immunity (Enjuanes et al., 2008; Yang et al., 2004). For SARS-CoV-2, studies with monoclonal antibodies have shown that SARS-CoV-2-infected humans develop robust neutralizing antibody responses against S and in particular the RBD (Baum et al., 2020; Hansen et al., 2020; Ju et al., 2020; Rogers et al., 2020; Shi et al., 2020; Zost et al., 2020).
In addition to S, early studies with SARS-CoV suggested that most infected individuals developed an antibody response to N (Pei et al., 2005).
N-protein-immunized BALB/c mice also induced CD4+ and CD8+ T cells (Liu et al., 2006). However, vaccination with vaccines expressing N resulted in no protection against SARS-CoV challenge as well as enhanced infection, which was characterized by increased pulmonary eosinophil infiltration (Deming et al., 2006).
Passive transfer of anti-N antibodies did not generate an enhanced response, leading the authors to believe that the increased severity was linked to a T cell response (Deming et al., 2006).
This was shown upon vaccination with VV expressing S, M, N, and E, as well as VV expressing N, causing the authors to trace back the response to nucleocapsid as an immunogen. Similarly, BALB/c mice vaccinated with a vaccinia virus (VV) expressing N showed enhanced viral infection upon SARS-CoV challenge, which was associated with a Th2 response with pulmonary infiltration of neutrophils, eosinophils, and lymphocytes (Yasui et al., 2008).
The M and E proteins have garnered less interest as vaccine targets due to lower immunogenicity (Du et al., 2008a), although SARS-CoV patient sera has been shown to be reactive to M peptides (Wang et al., 2003). Of the less-studied proteins, Orf3a has been shown to be capable of raising a neutralizing polyclonal antibody response in rabbits (Akerström et al., 2006).
Currently, Most Vaccines for SARS-CoV-2 Are Focused on S
To target the SARS-CoV-2 S protein in its native prefusion form, antigen stabilization strategies have been used (Figure 1 ). For MERS-CoV and SARS-CoV, introduction of two prolines in the S2 subunit effectively stabilized the S in its prefusion conformation (Pallesen et al., 2017).
The prefusion-stabilized MERS-CoV S generated higher neutralizing antibody titers when compared to native S trimer (Pallesen et al., 2017). This mutation prevented conformational changes of S in the presence of its receptor, ACE2, or trypsin (Kirchdoerfer et al., 2018).
This stabilization method has also been demonstrated to stabilize the SARS-CoV-2 S (Wrapp et al., 2020) and has been applied to multiple SARS-CoV-2 vaccine candidates (Corbett et al., 2020; Jackson et al., 2020; Mercado et al., 2020). Unlike SARS-CoV, but similar to MERS-CoV, SARS-CoV-2 includes a furin cleavage site between the S1 and S2 domains. For MERS-CoV, researchers mutated the furin cleavage site to enhance homogeneity and stability (Walls et al., 2019).
Some investigators have also added a foldon trimerization tag to the C terminus (Walls et al., 2020), and deletion of the cytoplasmic tail of the SARS-CoV S has been shown to increase neutralizing antibody titers (Yang et al., 2004). Current vaccine candidates in clinical trials are also exploring the inclusion of a tissue plasminogen activator leader sequence (tPA) with S (Folegatti et al., 2020; Mercado et al., 2020; Zhu et al., 2020a; Zhu et al., 2020).
The tPA leader can increase immunogen secretion and elicit increased humoral immune responses in influenza (Luo et al., 2008) and HIV vaccines (Wallace et al., 2013), although cellular immunogenicity was only increased in the influenza vaccine.
The RBD alone has also been explored as an immunogen. In preclinical studies in mice, rabbits, and monkeys, vaccination with modified vaccinia Ankara (MVA) expressing the full-length SARS-CoV S induced neutralizing antibodies that targeted RBD (Chen et al., 2005).
Moreover, depletion of RBD-specific antibodies significantly reduced convalescent plasma neutralizing capabilities (He et al., 2005), consistent with the concept that RBD is the primary target of S-specific neutralizing antibodies. When rabbits were immunized with a variety of SARS-CoV-2 S immunogens, including RBD, S1, S2, and modified variants, RBD was found to elicit five-fold higher affinity antibodies than the other immunogens (Ravichandran et al., 2020).
Sprague-Dawley rats immunized with SARS-CoV-2 RBD also elicited a strong neutralizing antibody response (Quinlan et al., 2020). RBD immunogens have been combined with a foldon trimerization tag in the BNT162b1 COVID-19 RNA vaccine candidate (Mulligan et al., 2020).
We recently compared a series of SARS-CoV-2 S immunogens in the context of both DNA vaccines and Ad26 vectors (Mercado et al., 2020; Yu et al., 2020). DNA vaccines encoding the full-length S elicited higher neutralizing antibody titers than did DNA vaccines encoding several S deletion mutants and also afforded improved protection against SARS-CoV-2 challenge in rhesus macaques (Yu et al., 2020).
Ad26 vectors were also generated encoding a series of S variants, and the full-length S with the PP-stabilizing mutations proved the most immunogenic and afforded the best protection against SARS-CoV-2 challenge in rhesus macaques (Mercado et al., 2020).
Preclinical Challenge Studies in NHPs
An optimal model for SARS-CoV-2 infection studies would involve an animal species permissive to viral replication and that develops pathologic and clinical features consistent with the human disease. Clinical manifestations of COVID-19 in humans are usually mild but can include cough, fever, pneumonia, and occasionally respiratory failure and death (Yang et al., 2020).
NHPs have significant genetic homology to humans and are often useful models for infectious diseases, although cost and availability can be limiting.
In both rhesus and cynomolgus macaques, virus shedding could be detected in nasal, throat, and rectal swabs and in bronchoalveolar lavage for approximately 2 weeks after infection with SARS-CoV-2 using either intratracheal and intranasal infection or intratracheal, intranasal, ocular, and oral infection (Chandrashekar et al., 2020; Munster et al., 2020; Rockx et al., 2020).
Early data suggest the utility of both macaque species as animal models for SARS-CoV-2 infection, although respiratory disease has been reported to be mild. The potential utility of African green monkeys as a model is also being explored. We recently demonstrated that SARS-CoV-2 infected macaques were also robustly protected against re-challenge, demonstrating natural protective immunity (Chandrashekar et al., 2020).
At least six SARS-CoV-2 vaccine challenge studies in macaques have been published at the time of this writing (Corbett et al., 2020; Gao et al., 2020; Mercado et al., 2020; van Doremalen et al., 2020; Wang et al., 2020; Yu et al., 2020) (Table 1 ). These vaccine studies in NHPs have included inactivated vaccines (PiCoVacc [Gao et al., 2020], BBIBP-CorV [Wang et al., 2020]), DNA vaccines (Yu et al., 2020), RNA vaccines (mRNA-1273 [Corbett et al., 2020]), and adenovirus-based vaccines (ChAdOx1 [van Doremalen et al., 2020], Ad26 [Mercado et al., 2020]).
PiCoVacc and BBIBP-CorV are based on SARS-CoV-2 CN2 and SARS-CoV-2 HB02 strains, respectively. The viruses were grown in Vero cells and inactivated by using β-propiolactone and were evaluated as two- or three-shot immunization regimens (Gao et al., 2020; Wang et al., 2020). The DNA and mRNA-1273 vaccines encoded stabilized S immunogens and were tested as two-shot immunization regimens (Yu et al., 2020; Corbett et al., 2020).
The ChAdOx1 vaccine expressed a codon-optimized full-length S with a human tPA leader sequence and was tested as a single-shot and a two-shot vaccine regimen (van Doremalen et al., 2020). The optimal Ad26 vaccine expressed a prefusion-stabilized S immunogen and was tested as a single-shot vaccine (Mercado et al., 2020).
NHP Challenge Studies of SARS-CoV-2 Vaccine Candidates
|Vaccine Name||Vaccine Type||Vaccine Immunogen||Vaccine Dose||Number of Injections||Challenge Route||Reference|
|PiCoVacc||inactivated||whole virus||3 or 6 μg||3||intratracheal||Gao et al., 2020|
|BBIBP-CorV||inactivated||whole virus||2 or 8 μg||2||intratracheal||Wang et al., 2020|
|DNA-S||DNA||engineered S||5 mg||2||intranasal and intratracheal||Yu et al., 2020|
|mRNA-1273||RNA||engineered S||10 or 100 μg||2||intranasal and intratracheal||Corbett et al., 2020|
|ChAdOx1 nCoV-19||adenoviral vector||tPA-S||2.5×1010 VP||1 or 2||intranasal, intratracheal, ocular, and oral||van Doremalen et al., 2020|
|Ad26.COV2.S (Ad26-S.PP)||adenoviral vector||engineered S||1×1011 VP||1||intranasal and intratracheal|
After vaccination, macaques were challenged by SARS-CoV-2 by the intratracheal, intranasal, oral, and/or ocular routes. Efficacy was determined in these studies by assessment of viral loads in the upper and lower respiratory tracts. Most studies are now focusing on analysis of subgenomic RNA rather than genomic RNA (Wölfel et al., 2020), because subgenomic RNA is believed to be more reflective of replicating virus, rather than input challenge virus.
The inactivated vaccine PiCoVacc resulted in reduced viral loads in throat swabs (Gao et al., 2020). The inactivated vaccine BBIBP-CorV also decreased viral loads in throat swabs (Wang et al., 2020). The DNA vaccine encoding the full-length S resulted in >3.1 and >3.7 log10 reductions in median subgenomic RNA levels in BAL and nasal swabs, respectively (Yu et al., 2020).
The mRNA-1273 vaccine resulted in undetectable subgenomic RNA in lungs in all but one macaque, and more rapid clearance of virus from nasal swabs (Corbett et al., 2020). The optimal Ad26-S.PP vaccine resulted in undetectable subgenomic RNA in lungs and only one breakthrough in nasal swabs (Mercado et al., 2020). The ChAdOx1 vaccine resulted in reduced subgenomic RNA in BAL but no decrease in nasal swabs (van Doremalen et al., 2020).
The different challenge doses, strains, routes, and assays that were utilized make direct comparisons among these studies difficult. Nevertheless, these studies provide a substantial amount of preclinical data demonstrating protective efficacy of multiple vaccine candidates in NHPs. These data help inform the ongoing clinical development programs for these vaccines.
A limitation of all these studies is that macaques do not develop severe disease, respiratory failure, or death. Small animal models are currently being developed that could model severe disease, including hamsters and transgenic mice.
Immune Correlates of Protection
Determining immune correlates of protection (CoP) for SARS-CoV-2 will be critical for guiding vaccine development. Currently, mechanistic CoP for SARS-CoV-2 have not yet been fully determined, although several studies point to the importance of both humoral and cellular immunity.
Neutralizing antibodies (nAbs) represent a commonly studied immune correlate of protection. Early human challenge studies reported that volunteers with higher pre-existing anti-CoV 229E nAb titers (> 5) demonstrated lower proportions of virus isolation and upper respiratory infection than those with low neutralizing titers (≤5) (Bradburne et al., 1967).
Pre-challenge serum antibody titers were also negatively associated with upper respiratory infections, nasal virus shedding, and disease severity (Barrow et al., 1990; Callow, 1985). Passive administration of convalescent sera, purified IgG, or monoclonal antibodies have also been shown to suppress SARS-CoV challenge/infection and associated disease progression in mice, hamsters, ferrets, and humans (Cheng et al., 2005; Roberts et al., 2006; Subbarao et al., 2004; Sui et al., 2005; Yuan et al., 2015).
For SARS-CoV-2, neutralizing monoclonal antibodies isolated from convalescent COVID-19 patients have been shown to inhibit SARS-CoV-2 infection in both prophylactic and therapeutic settings in rhesus macaques and hamsters (Rogers et al., 2020; Shi et al., 2020; Zost et al., 2020).
We also reported that DNA vaccines and Ad26-based vaccines induced nAbs that strongly correlated with a reduction of viral loads in rhesus macaques (Mercado et al., 2020; Yu et al., 2020). SARS-CoV-2 inactivated virus vaccines and mRNA vaccines also induced nAbs and conferred protection in macaques (Corbett et al., 2020; Gao et al., 2020; Wang et al., 2020).
Collectively, these studies suggest that nAb titers could serve as a useful biomarker for evaluating SARS-CoV-2 vaccines in both preclinical and clinical studies, although these correlates need to be confirmed in humans.
A question that has been raised is whether sub-neutralizing levels of antibodies could have detrimental effects. A prior study revealed that a vaccinia vector-based vaccine expressing feline coronavirus S induced low titers of neutralizing antibodies, which led to early cat mortality upon challenge (Vennema et al., 1990).
Consistent with this observation, several studies observed enhanced respiratory disease (ERD) in response to SARS vaccines when antibodies had suboptimal potency or low binding affinity (Jaume et al., 2011; Wan et al., 2020; Wang et al., 2014). A major effort in the field will therefore be the development of animal models for ERD for SARS-CoV-2.
In addition to neutralization, emerging evidence suggests that certain antibody Fc-mediated functionscould also contribute to protective efficacy (Yu et al., 2020), including antibody-dependent complement deposition (ADCD), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent NK cell activation (ADNKA) (Zohar and Alter, 2020).
Mucosal immunity is also likely important for protection, because coronavirus infection occurs in the respiratory tract and potentially in the gastrointestinal tract (Xiao et al., 2020). Pulmonary immunoglobulin (Ig)A was observed to be inversely associated with MERS-CoV infectivity in humans (Muth et al., 2015), and animal studies have highlighted the potential role of mucosal immunity in defending SARS-CoV infection (Du et al., 2008b; Huang et al., 2009).
Given that intranasal administration could induce stronger mucosal immunity in the respiratory tract than parenteral routes, it could prove useful to evaluate intranasal routes for vaccines (Roper and Rehm, 2009).
Cellular immunity also appears important in the control of coronavirus infections. Current evidence suggests that not all patients develop protective humoral immune responses, and asymptomatic patients or individuals with mild disease typically develop robust T cell responses (Mathew et al., 2020; Sekine et al., 2020).
In a mouse model, SARS-CoV-specific CD8+ T cell numbers correlated with virus clearance and increased survival (Zhao et al., 2010). In addition, memory CD4+ T cells were associated with protective immunity against MERS-CoV (Zhao et al., 2016). Recent studies have reported that neutralizing antibody titers correlated with SARS-CoV-2-specific T cell responses (Ni et al., 2020) and that IgG and IgA antibody titers correlated with S-specific CD4+ T cell responses (Grifoni et al., 2020), suggesting that T cells could indirectly modulate the virus infection by orchestrating antibody production.
However, in DNA vaccine studies in mice and NHPs, T cell responses did not correlate with protection (Yang et al., 2004; Yu et al., 2020). Nevertheless, severe patients tend to have a higher frequency of polyfunctional CD4 cells expressing interferon (IFN)γ, interleukin (IL)-2, and tumor necrosis factor alpha (TNF-α) (Sekine et al., 2020; Thieme et al., 2020), although it is possible that these high-frequency T cells could simply represent long-term viral exposure (Altmann and Boyton, 2020).
There is limited data on innate immune correlates of protection. Existing pathogenesis studies suggest that the excessive production of proinflammatory cytokines and chemokines (cytokine storm) is associated with poor clinical outcome (Channappanavar and Perlman, 2017). IFN-I and IFN-III have shown to be able to suppress SARS-CoV-2 infection in vitro (Sallard et al., 2020; Stanifer et al., 2020) and could represent innate immune responses that assist in controlling SARS-CoV-2 infection.
Blocking IFN-I signaling in mice has been shown to increase viral load and mortality for SARS-CoV and MERS-CoV (Channappanavar et al., 2019; Frieman et al., 2010). Retrospective cohort studies have also suggested that induction of IFNs correlated with disease severity and viral load in both SARS and MERS patients (Cameron et al., 2007; Kim et al., 2016). It is possible that early induction promotes viral clearance, whereas delayed IFN responses can cause viral persistence, inflammation, and immunopathology (Park and Iwasaki, 2020).
The potential off-target effects of BCG, historically used as a vaccine for tuberculosis, and other live attenuated organisms are also being explored for SARS-CoV-2 (Curtis et al., 2020). Trained immunity is regarded as immunological memory in the innate immune system and likely mediated by epigenetic modifications (Netea et al., 2016). If non-specific trained innate immunity protects against SARS-CoV-2, this would provide an intriguing alternate to antigen-specific vaccination.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416703/
More information: Noe B. Mercado et al, Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques, Nature (2020). DOI: 10.1038/s41586-020-2607-z