Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals (preprint)

Background: As there are limited data on B cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Methods: Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n=30), WT infected (n=30), delta infected (n=30), omicron infected+vaccinated (n=20) and Sinopharm (BBIBP-CorV) vaccinees (n=30). We then investigated the sensitivity and specificity of these immunodominant regions and analysed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses and bat Sarbecoviruses. We then investigated the kinetics of responses to these regions in those with varying severity of acute COVID-19. Results: We identified four immunodominant regions aa 29-52, aa 155-178, aa 274 to 297 and aa 365 to 388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, with WT infected individuals predominantly recognizing aa155 to 178 regions, delta infected individuals and vaccinated+omicron infected individuals predominantly recognizing regions aa 29 to 52 and aa 274 to 294 regions. Sinopharm vaccinees recognized all four regions, with the magnitude of responses significantly lower than other groups. >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. Conclusions: N-protein specific responses appear to be detectable in over 90% of those who were naturally infected or vaccinated with a whole virus inactivated vaccine, with responses mainly directed against four regions of the protein, which were highly conserved. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

A randomized control trial to assess the efficacy and safety of ivermectin in the treatment of mild to moderate COVID 19 patients

Comparison of kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222 (preprint)

Background While there have been many studies characterizing the IgG and IgA responses to different SARS-CoV-2 proteins in individuals with natural infection, the induction of IgG and IgA to different viral proteins in vaccinees have not been extensively studied. Therefore, we sought to investigate the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD2221, in Sri Lankan individuals. Methods Using Luminex assays, we characterized the IgG and IgA responses in patients with varying severity of illness and following a single dose of the vaccine at 4 weeks and 12 weeks since onset of illness or following vaccination. Haemagglutination test (HAT) was used to assess the antibodies to the receptor binding domain of SARS-CoV-2 wild type (WT), B.1.1.7, B.1.351 and B.1.617.2 (VOCs) and surrogate neutralizing test to measure ACE2 receptor blocking antibodies. Results Those with mild illness and in vaccinees, the IgG responses to S1, S2, RBD and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. Those who had a febrile illness in 2017 and 2018 (controls) also gave IgG and IgA high responses to the S2 subunit. In the vaccinees, the most significant rise was seen for the IgG antibodies to the S2 subunit (p<0.0001). Vaccinees had several fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with mild and moderate/severe illness at 4 weeks and 12 weeks. At 12 weeks the HAT titres were significantly lower to the B.1.1.7 in vaccinees and significantly lower in those with mild illness, and in vaccinees to B.1.351 and for B.1.617.2. No such difference was seen in those with moderate/severe illness. Conclusions Vaccinees had significantly less IgA to SARS-CoV-2, but comparable IgG responses to those with natural infection. However, following a single dose, vaccinees had reduced antibody levels to the variants of concern (VOC), which further declined with time, compared to natural infection.

Genomic and epidemiological analysis of SARS-CoV-2 viruses in Sri Lanka (preprint)

Since identification of the first Sri Lankan individual with the SARS-CoV-2 in early March 2020, small clusters that occurred were largely contained until the current extensive outbreak that started in early October 2020. In order to understand the molecular epidemiology of SARS-CoV-2 in Sri Lanka, we carried out genomic sequencing overlaid on available epidemiological data. The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country. The estimated time of the most recent common ancestor of this lineage was 10th August 2020 (95% lower and upper bounds 6th July to 7th September), suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258 , as well as the more transmissible B.1.1.7 lineage. Ongoing genomic surveillance in Sri Lanka is vital as vaccine roll-out increases.

SARS-CoV-2 neutralizing antibodies in patients with varying severity of acute COVID-19 illness (preprint)

In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points.Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p=0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had Nabs, 38/76 (50%) in those with >90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (>90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness.

Similarities and differences between the ‘cytokine storms’ in acute dengue and COVID-19 (preprint)

Severe pneumonia and multiorgan dysfunction in COVID-19 and dengue haemorrhagic fever (DHF) are two diseases that can associate with an altered immune response to the infecting virus. To determine the similarities and differences in the cytokine and chemokine responses in these two infections, we compared responses in patients with varying severity of COVID-19 and acute dengue at different time points of illness.During early disease, patients who proceeded to develop COVID-19 severe pneumonia (SP) and DHF had significantly higher levels of IL-6, IL-10 and MIP3α than those who developed mild illness. The lowest levels of IFNγ in early illness were seen in those who succumbed to their illness due to COVID-19. Levels of serum IL-10 (p=0.0001), IL-6 (p=0.002), MIP-3α (p=0.02) and CD40-L levels (p=0.002) significantly increased from 5-9 day of illness to 10-21 day of illness in patients with moderate-to-severe COVID-19, but not in those with mild illness. In contrast, these cytokine/chemokine levels remained unchanged in those with DHF or dengue fever (DF) during febrile and critical phases. Although IL-10 levels were significantly higher in COVID-19 patients with SP, patients with DHF had 25-fold higher levels, whereas IL-6 levels were 11-fold higher in those with COVID-19 SP. IL-10 and other cytokines were evaluated in a larger cohort of patients during early illness (≤ 4 days) who proceeded to develop DF (n=71) or DHF (n=64). Of the cytokines evaluated, IL-10 was significantly higher (p<0.0001) in those who went on to develop DHF compared to DF. Low IFNγ response to the SARS-CoV2 and high levels of immunosuppressive IL-10 in both COVID-19 and dengue during early illness are indicators of an altered antiviral response potentially contributing to disease severity.