ABSTRACT
The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid (Poly(I:C)) adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after one or two boosts. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation (spike protein with Alum + Poly(I:C)) in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.
ABSTRACT
Prolonged infection of SARS-CoV-2 represents a challenge to the development of effective public health policies to control the COVID-19 pandemic. The reason why some people have persistent infection and how the virus survives for so long are still not fully understood. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during persistent infection. Thirty-three patients who remained RT-PCR positive in the nasopharynx for at least 16 days were included in this study. Complete SARS-CoV-2 sequences were obtained for each patient at two time points. Phylogenetic, populational, and computational analysis of viral sequences confirmed persistent infection with evidence for a transmission cluster in health care professionals that shared the same workplace. A high number of missense variants targeting crucial structural and non-structural proteins such as Spike and Helicase was found. Interestingly, longitudinal acquisition of substitutions in Spike protein mapped many SARS-CoV-2 predicted T cell epitopes. Furthermore, the mutational profiles observed were suggestive of RNA editing enzyme activities, indicating innate immune mechanisms of the host cell. Viral quasispecies analysis corroborates persistent infection mainly by increasing richness and nucleotide diversity over time. Altogether, our findings highlight a dynamic and complex landscape of host and pathogen interaction during persistent infection suggesting that the hosts innate immunity shapes the increase of intra-host diversity with possible implications for therapeutic strategies and public health decisions during the COVID-19 pandemic.
ABSTRACT
Accurate serological tests are essential tools to allow adequate monitoring and control of COVID-19 spread. Production of a low-cost and high-quality recombinant viral antigen can enable the development of reliable and affordable serological assays, which are urgently needed to facilitate epidemiological surveillance studies in low-income economies. Trimeric SARS-COV-2 spike (S) protein was produced in serum-free, suspension-adapted HEK293 cells. Highly purified S protein was used to develop an ELISA, named S-UFRJ test. It was standardized to work with different types of samples: (i) plasma or serum from venous blood samples; (ii) eluates from dried blood spots (DBS) obtained by collecting blood drops from a finger prick. We developed a cost-effective, scalable technology to produce S protein based on its stable expression in HEK293 cells. Using this recombinant antigen, we presented a workflow for test development in the setting of a pandemic, starting from limited amounts of samples up to reaching final validation with hundreds of samples. Test specificity was determined to be 98.6%, whereas sensitivity was 95% for samples collected 11 or more days after symptoms onset. A ROC analysis allowed optimizing the cut-off and confirming the high accuracy of the test. Endpoint titers were shown to correlate with virus neutralization assessed as PRNT90. There was excellent agreement between plasma and DBS samples, significantly simplifying sample collection, storing, and shipping. An overall cost estimate revealed that the final retail price could be in the range of one US dollar. The S-UFRJ assay developed herein meets the quality requirements of high sensitivity and specificity. The low cost and the use of mailable DBS samples allow for serological surveillance and follow-up of SARS-CoV-2 vaccination of populations regardless of geographical and socio-economic aspects. We hope the detailed guidelines for the development of an affordable and accurate anti-spike SARS-COV-2 ELISA, such as S-UFRJ described here, will stimulate governmental and non-governmental health agencies in other countries to engage in much-needed large-scale studies monitoring the spread and immunity to SARS-COV-2 infection.
