The ongoing pandemic of COVID-19 has taken a huge health, economic, and social toll on humanity. To restore some semblance of normalcy, scientists have been focusing on finding effective vaccines and antivirals. A new report by researchers at the University of Melbourne and published on the preprint server bioRxiv* in September 2020 reports on the use of a two-protein two-dose vaccine regimen and its immunogenicity in two animal models.
Spike-Based Neutralization
The spike (S) glycoprotein of SARS-CoV-2 has been the focus of many attempts to design novel vaccines to elicit neutralizing antibodies. Prior research shows that these antibodies can prevent infection in macaques and reinfection in humans.
The spike protein mediates viral attachment with the angiotensin-converting enzyme 2 (ACE2) enzyme on the host cell surface, with subsequent fusion and viral entry into the cell. This engagement occurs at the receptor-binding domain (RBD). Antibodies that prevent the RBD-ACE2 binding are, therefore, an efficient route to the neutralization of the virus.
Molecule of SARS-CoV-2 coronavirus main protease, 3D illustration. Image Credit: Kateryna Kon / Shutterstock
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Viruses also evade the immune system by a host of methods, including heavy N-glycosylation, exposing viral regions which constantly change to the immune system, and other techniques. The vaccine development protocols must ensure that most B cells recognized those epitopes that are most crucial for viral replication, or on-target epitopes, leaving those which are mutable or non-neutralizing (off-target epitopes).
While multiple spike-based vaccines are now in trials, they use various spike presentations such as recombinant spike trimers, RBD protomers or trimers, and spike analogs that enter the cell via viral vectors or mRNAs. The variety of vaccine candidates shows that many questions remain about the optimal immunogen to be use to achieve the most targeted neutralization response.
Especially should the vaccine include only the RBD, which binds to the host receptor, or larger sections of the spike, including many more epitopes, to increase immunogenicity and neutralization potential? Or on the other hand, will this only increase off-target epitopes and fall prey to the immune distraction strategy of the virus?
S or RBD: Which is Better?
The current study compares the immune response elicited by the S and RBD epitopes in mice and nonhuman primates using a two-dose regimen in various forms. The first primes the immune system while the second re-challenges it and ensures effective antibody concentrations.
The researchers found that in mice, the RBD is a poor immunogen, being unable to induce significant B cell responses in the germinal centers (GC), or to recruit T follicular helper cells (Tfh). When S alone is used as the prime dose, followed by either S or RBD boost, the immune response is robust, with both binding and neutralizing antibodies being produced.
In nonhuman primates, on the other hand, a prime-boost regimen with either of these proteins was reliably found to induce strong neutralizing antibody responses. Recombinant S immunogens were selected for further study as they elicit a protective antibody response at a level higher than that seen in convalescent humans.
Spike but Not RBD Powerfully Immunogenic in Mice
A single dose of the adjuvanted spike protein, independent of glycosylation, was found to produce high anti-S antibodies with one dose, but had low neutralizing capacity. One dose of RBD was not very immunogenic, as expected.
GC B cells targeting both S and RBD were induced robustly by S but not by RBD.
Tfh cells showed the same trend, with the predominant immunogenic epitopes being non-RBD. Only 3 peptide sequences on the RBD were recognized by CD4 T cells, one being much more immunogenic than the others.
The S protein had over 8 non-RBD epitopes outside the RBD, which were more immunogenic than the RBD epitopes and induced a more potent CD4 T cell response.
The S-R regime led to a fourfold rise in binding and neutralizing antibodies against the RBD relative to a homologous S-S immunization. When the spike protein was first used, followed by an RBD boost to focus the antibodies on the RBD, the neutralizing activity was 2.5 times higher, in contrast to an S-S protocol. The S protein is more potent in eliciting an antibody response independent of the boost antigen in a prime-boost regime, directing GC B and Tfh cell responses to non-RBD epitopes. On the contrary, the R-R or R-S regimes were poorly immunogenic for antibodies against RBD or S.
The R-R also produced the lowest GC B cell responses, intermediate responses being achieved with the use of the spike as the priming dose and either spike or RBD as the booster, and robust induction with R-S immunization in mice, probably because the spike protein elicits GC B cell responses against non-RBD epitopes on the spike protein.
RBD and S Show Potent Antibody and Neutralizing Activity in Non-Human Primates
The researchers recapitulated their findings in a nonhuman primate (NHP) model, the pig-tailed macaque, using both RBD and S protein vaccines. They found that two doses of homologous or heterologous vaccine reliably induced neutralizing activity against both proteins, without the variation in immunogenicity observed in mice
GC B/Tfh cell responses were robust in all animals, but higher with S-S and S-R compared to R-R. Specific GC B cell induction targeting the RBD was highest in R-R animals, followed by S-R and S-S. However, anti-S B cells were highest in S-S and S-R, but low in R-R immunized animals.
GC Tfh cells were found in all draining lymph nodes, targeting both RBD and non-RBD peptides, but the Tfh more often recognized the former.
Memory B cells specific for S and RBD epitopes are crucial for long-lasting immunity. S-S immunization led to the highest frequencies of S-specific memory B cells, with R-R and S-R leading to comparable frequencies.
RBD-targeting B cells were low throughout, but highest among S-S animals with lower levels in S-R and R-R groups. After two weeks of vaccine boost, both memory CD4 T cells and Tfh cells were found in circulation.
A surprising finding was that S-targeting circulating Tfh targeted both RBD and non-RBD peptides equally in NHPs, the former corresponding to the S antibody titers. This shows that possibly, “antigen-specific cTFH constitute a useful biomarker of vaccine immunogenicity in NHP models.”
Immune Responses in Mice, Macaques and Humans
The researchers then evaluated how well S and RBD induce antibodies and the neutralizing titer in 72 COVID-19 convalescent plasma samples. They found that compared to natural human infection, sera from mice and NHPs immunized with S-S and S-R have high antibody and neutralizing activity. In the latter, the neutralizing activity was above that found in convalescent human plasma.
They also found that unlike in animals, humans had specific RBD and S-specific cells from multiple V gene families. When it comes to the length of CDR277 H3, they found that this was shorter in mice than in macaques (either from the lymph nodes or peripheral blood mononuclear cells (PBMCs). The macaque CDR277 H3 lengths were comparable to those found in convalescents, for both RBD and S sequences.
Implications
The current study displays the benefit of combining immunogens and comparing their performance in different animal models and in humans. As reported earlier, RBD has lower immunogenicity in mice after either one or two doses because of the inadequate recruitment of Tfh of high efficacy as part of the primary immune response.
However, S priming in mice was found to be a potent inducer of the primary binding and neutralizing antibody response, independent of the boost antigen. This shows that priming a broader spectrum of Tfh cells helps to regulate the immune response after boosting. Moreover, RBD was found to be more immunogenic in NHPs because of their greater allelic and MHC II repertoire, compared to mice.
Still, RBD is not well recognized by either GC B or circulating Tfh cells in COVID-19 convalescents, which makes it probably a less-preferred immunogen at the population level. The spike antigen was much more immunogenic in all species.
The use of spike priming with heterologous RBD boosting caused antibody recognition to focus on the ACE2 recognition site in mice, indicating selective recall of RBD response with increased inhibition of RBD-ACE2 binding. This difference was not seen in macaques immunized with S-R vs. S-S, reflecting the human response, as recently shown by the same team.
The researchers comment, “Serological neutralizing activity was not exclusively RBD-directed.” Instead, the N-terminal domain (NTD) and other non-RBD epitopes may be responsible for much of the vaccine protection. Secondly, mice use different gene families and have limits on the CDR H3 lengths in their GC B cell responses to vaccines. This distinguishes them from NHP or human subjects and is possibly the reason why they require high antibody titers for neutralization compared to the latter.
Recent research suggests that human monoclonal antibodies that neutralize non-RBD epitopes have longer CDR-H3 loops than seen in mice, and this may influence the antibody response to spike-based vaccines in mice. Accordingly, changes may be envisaged in the design of similar vaccines in humans.
Finally, the weak and transient neutralizing antibody titers following natural SARS-CoV-2 infection in humans may be countered by a simple adjuvanted two-protein prime-boost regime. The response included robust GC B cell induction with specific anti-S activity, as well as memory T cells and B cells in peripheral blood. The study concludes, “Vaccination constitutes a more robust and reliable pathway to serological protection against SARS-CoV-2 than natural infection.”
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- Mar 30 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
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