The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80–100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25–250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, a plethora of ultraviolet-C (UV-C) disinfection products have come to market, especially in emerging economies. UV-C–based disinfection products for mobile phones, food packaging, face masks and personal protective equipment (PPE), and other everyday objects are available in popular electronic-commerce platforms as consumer products. Product designers from multinational to startup companies began to design UV-C disinfection products but had no prior-art reference, user feedback, or validation of product efficacy, which are important stages in product design. A UV-C disinfection product cannot be assessed by most consumers for its viricidal efficacy. Many firms entered the domain of UV-C products and were unaware of the necessary validation requirements. Lack of availability and access to virology laboratories, due to lockdowns in countries, and lack of standards and certification for UV-C disinfection products limited product designers and firms in benchmarking their UV-C–based devices before market release. This work evaluates two UV-C disinfection devices for viricidal efficacy on PPE fabric and National Institute for Occupational Safety and Health (NIOSH)–certified N95 respirators through controlled experiments using the H1N1 virus, which is enveloped and is transmitted via the respiratory route similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19. The experiment also evaluated the effectiveness of chemical disinfectants along with and versus UV-C disinfection. Experiments for material selection, UV dose calculation, and UV endurance of PPE samples to be disinfected are also discussed. The outcome of this work establishes a systematic method to validate the efficacy of UV-C disinfection products. The design guidelines would benefit product designers in designing UV-C–based disinfection products.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD. The protein is highly thermotolerant and immunogenic and is being explored for use in room temperature stable Covid-19 vaccine formulations. In the current study, we have investigated the folding pathway of both WT and stabilized RBD. It was found that chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Thermal and chemical denaturation is reversible, as assayed by binding to the receptor ACE2. Unusually, in its native state, RBD binds to the hydrophobic probe ANS, and enhanced ANS binding is observed for the equilibrium intermediate state. Further characterization of the folding of mRBD1-3.2, both in solution and after reconstitution of lyophilized protein stored for a month at 37 °C, revealed a higher stability represented by higher C_m, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. In contrast to WT RBD, the mutant showed decreased interaction with the hydrophobic moiety linoleic acid. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system.

During the coronavirus disease 2019 (COVID-19) pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here, we report an analysis of 164 serum samples isolated from patients with breakthrough infection in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wild-type (WT), Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping, using charged scanning mutagenesis coupled with yeast surface display and next-generation sequencing. The mapped sera primarily targeted the recently identified class 5 cryptic epitope and, to a lesser extent, the class 1 and class 4 epitopes. The class 5 epitope is completely conserved across all severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and for most sarbecoviruses. Based on these observations, an additional 26 sera were characterized, and all showed a broad neutralizing activity, including against XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variants of concern (VOCs) were observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population were sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern. These data highlight the important role of hybrid immunity in conferring broad protection and inform future vaccine strategies to protect against rapidly mutating viruses. IMPORTANCE: Worldwide implementation of coronavirus disease 2019 (COVID-19) vaccines and the parallel emergence of newer severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have shaped the humoral immune response in a population-specific manner. While characterizing this immune response is important for monitoring disease progression at the population level, it is also imperative for developing effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the world’s second largest COVID-19 vaccination drive and encountered a large number of post-vaccination “breakthrough” infections. From a cohort of patients with breakthrough infection, we identified individuals whose sera showed broadly neutralizing immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope, which was not identified in previous population-level studies conducted in Western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants, including recently emerged ones and for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

Background: Adjuvants function by enhancing the breadth, durability, and magnitude of the immune response, but little is known about their impact on vaccine stability. CpG is a widely used adjuvant that is included in several recently approved COVID-19 vaccines using Spike protein, RBD, or whole inactivated virus. Methods: Here, we investigate the in vitro stability of the Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein, as well as a number of other proteins formulated with a class B CpG adjuvant. Results: We show that RBD, BSA, and lysozyme proteins are less thermally stable, more aggregation-prone, and more protease-sensitive in the presence of CpG than without it, and that these effects are enhanced with prolonged incubation. For RBD, the effects of CpG are pH-independent but dependent on the salt concentration, with relative destabilisation decreasing with an increasing salt concentration, indicative of an electrostatic component to the interaction between CpG and the protein. The reduced thermal and proteolytic stability found in the presence of CpG is indicative of a preferential interaction of CpG with the unfolded state of the protein relative to its native state. It remains to be determined if these in vitro characteristics are unique to CpG or are also shared by other non-CpG commercial adjuvants, if they are antigen-dependent, and if and how they correlate with the in vivo immunogenicity of an adjuvanted vaccine. Conclusions: It is demonstrated that the CpG adjuvant is critical to enhancing immunogenicity and is a key reason for the success of multiple licensed commercial vaccines. Nonetheless, our work suggests that careful and systematic in vitro formulation studies may be warranted for the development of suitable, stable formulations of CpG-adjuvanted vaccine candidates.