Immunogenicity Evaluation of Thermostable Microparticles Entrapping Receptor Binding Domain of SARS-CoV-2 by Single Point Administration.

Receptor binding domain (RBD) of SARS-CoV-2 is a prime vaccine target against which neutralizing antibody responses are directed. Purified RBD as a vaccine candidate warrants administration of multiple doses along with adjuvants and use of delivery systems to improve its immunogenicity. The present investigation examines the immunogenicity of RBD delivered by biodegradable polymer particles from single dose administration. Mice upon single point immunization of RBD entrapped microparticles generated improved antibody response. The polymer microparticles showed better temperature stability and could be stored at 37 degrees for one month without any considerable loss of immunogenicity. Further, immunization with microparticles could elicit memory antibody response upon challenge after four months of single dose administration. Thus, using microparticles entrapping RBD as a vaccine candidate confer improved immunogenicity, temperature stability and recall response. These thermostable microparticles seem to be a potentially cost-effective approach which can help in dose reduction, provide a wider access of vaccines and accelerate the end of global pandemic.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

Thermostable Vaccines in Veterinary Medicine: State of the Art and Opportunities to Be Seized.

The COVID-19 pandemic has highlighted the weakness of the vaccine supply chain, and the lack of thermostable formulations is one of its major limitations. This study presents evidence from peer-reviewed literature on the development of thermostable vaccines for veterinary use. A systematic review and meta-analysis were performed to evaluate the immunogenicity and/or the efficacy/effectiveness of thermostable vaccines against infectious diseases. The selected studies (n = 78) assessed the vaccine's heat stability under different temperature conditions and over different periods. Only one study assessed the exposure of the vaccine to freezing temperatures. Two field studies provided robust evidence on the immunogenicity of commercial vaccines stored at temperatures far in excess of the manufacturer's recommended cold-chain conditions. The drying process was the most-used method to improve the vaccine's thermostability, along with the use of different stabilizers. The pooled vaccine efficacy was estimated to be high (VE = 69%), highlighting the importance of vaccination in reducing the economic losses due to the disease impact. These findings provide evidence on the needs and benefits of developing a portfolio of heat- and freeze-stable veterinary vaccines to unleash the true potential of immunization as an essential component of improved animal health and welfare, reduce the burden of certain zoonotic events and thus contribute to economic resilience worldwide.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

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.

Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment.

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.

The Development of a Standardized Quality Assessment Material to Support Xpert® HIV-1 Viral Load Testing for ART Monitoring in South Africa.

The tiered laboratory framework for human immunodeficiency virus (HIV) viral load monitoring accommodates a range of HIV viral load testing platforms, with quality assessment critical to ensure quality patient testing. HIV plasma viral load testing is challenged by the instability of viral RNA. An approach using an RNA stabilizing buffer is described for the Xpert® HIV-1 Viral Load (Cepheid) assay and was tested in remote laboratories in South Africa. Plasma panels with known HIV viral titres were prepared in PrimeStore molecular transport medium for per-module verification and per-instrument external quality assessment. The panels were transported at ambient temperatures to 13 testing laboratories during 2017 and 2018, tested according to standard procedures and uploaded to a web portal for analysis. A total of 275 quality assessment specimens (57 verification panels and two EQA cycles) were tested. All participating laboratories met study verification criteria (n = 171 specimens) with an overall concordance correlation coefficient (ρc) of 0.997 (95% confidence interval (CI): 0.996 to 0.998) and a mean bias of -0.019 log copies per milliliter (cp/mL) (95% CI: -0.044 to 0.063). The overall EQA ρc (n = 104 specimens) was 0.999 (95% CI: 0.998 to 0.999), with a mean bias of 0.03 log cp/mL (95% CI: 0.02 to 0.05). These panels are suitable for use in quality monitoring of Xpert® HIV-1 VL and are applicable to laboratories in remote settings.