K. Rungrojcharoenkit’s research while affiliated with Armed Forces Research Institute of Medical Sciences and other places

Setting up a global SARS-CoV-2 surveillance system requires an understanding of how virus isolation and propagation practices, use of animal or human sera, and different neutralisation assay platforms influence assessment of SARS-CoV-2 antigenicity. In this study, with the contribution of 15 independent laboratories across all WHO regions, we carried out a controlled analysis of neutralisation assay platforms using the first WHO International Standard for antibodies to SARS-CoV-2 variants of concern (source: NIBSC). Live virus isolates (source: WHO BioHub or individual labs) or spike plasmids (individual labs) for pseudovirus production were used to perform neutralisation assays using the same serum panels. When comparing fold drops, excellent data consistency was observed across the labs using common reagents, including between pseudovirus and live virus neutralisation assays (RMSD of data from mean fold drop was 0.59). Utilising a Bayesian model, geometric mean titres and assay titre magnitudes (offsets) can describe the data efficiently. Titre magnitudes were seen to vary largely even for labs within the same assay group. We have observed that overall, live Microneutralisation assays tend to have the lowest titres, whereas Pseudovirus Neutralisation have the highest (with a mean difference of 3.2 log2 units between the two). These findings are relevant for laboratory networks, such as the WHO Coronavirus Laboratory Network (CoViNet), that seek to support a global surveillance system for evolution and antigenic characterisation of variants to support monitoring of population immunity and vaccine composition policy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is found in regions where dengue (DENV) and chikungunya (CHIKV) viruses are endemic. Any serological cross-reactivity between DENV, CHIKV, and SARS-CoV-2 is significant as it could lead to misdiagnosis, increased severity, or cross-protection. This study examined the potential cross-reactivity of anti-DENV and CHIKV antibodies with SARS-CoV-2 using acute and convalescent-phase samples collected before the SARS-CoV-2 pandemic. These included healthy, normal human (NHS, n = 6), CHIKV-positive (n = 14 pairs acute and convalescent), primary DENV-positive (n = 20 pairs), secondary DENV-positive (n = 20 pairs), and other febrile illnesses sera (n = 23 pairs). Samples were tested using an in-house SARS-CoV-2 and a EUROIMMUN IgA and IgG ELISAs. All NHS samples were negative, whereas 3.6% CHIKV, 21.7% primary DENV, 15.7% secondary DENV, and 10.8% febrile diseases sera resulted as anti-SARS-CoV-2 antibody positive. The EUROIMMUN ELISA using spike 1 as the antigen detected more positives among the primary DENV infections than the in-house ELISA using spike 1-receptor binding domain (RBD) protein. Among ELISA-positive samples, four had detectable neutralizing antibodies against SARS-CoV-2 reporter virus particles yet none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated the SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. IMPORTANCE SARS-CoV-2 continues to cause significant morbidity globally, including in areas where DENV and CHIKV are endemic. Reports using rapid diagnostic and ELISAs have demonstrated that serological cross-reactivity between DENV and SARS-CoV-2 can occur. Furthermore, it has been observed that convalescent DENV patients are at a lower risk of developing COVID-19. This phenomenon can interfere with the accuracy of serological testing and clinical management of both DENV and COVID-19 patients. In this study, the cross-reactivity of primary/secondary anti-DENV, CHIKV, and other febrile illness antibodies with SARS-CoV-2 using two ELISAs has been shown. Among ELISA-positive samples, four had detectable levels of neutralizing antibodies against SARS-CoV-2 reporter virus particles. However, none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. The data discussed here provide information regarding diagnosis and may help guide appropriate public health interventions.

This study measures the impact of chloroquine (CQ) therapy in reducing SARS-CoV-2 viral load in infected individuals and hence its transmissibility by describing changes in nasopharyngeal SARS-CoV-2 RNA kinetics in patients receiving standard of care (SOC) or CQ +/-ritonavir-boosted lopinavir (LPV/r). The nasopharyngeal (NP) samples were collected from mild COVID-19 patients admitted at Bamrasnaradura Infectious Diseases Institute between March and April of 2020. These patients either received SOC, or a high dose of CQ with loading dose, or high dose of CQ plus LPV/r. The samples were tested at AFRIMS using a quantitative RT-PCR assay. Levels of CQ in the plasma were measured 6 days post initiation of their treatment. In some instances, viral isolation was attempted to determine SARS-CoV-2 viability. Analyses of the

Background We compared cord blood antibody titers in unvaccinated pregnant women to those vaccinated with seasonal influenza vaccine during the 2nd and the 3rd trimesters. Methods Pregnant women had cord blood collected at delivery for hemagglutination inhibition assay against vaccine reference viruses: A/California/07/2009 (H1N1)pdm09, A/Switzerland/9715293/2013 (H3N2), and B/Phuket/3073/2013 (Yamagata lineage). Geometric mean titer (GMT) ratios were calculated comparing vaccinated versus unvaccinated pregnant women, and women vaccinated in the 2nd and the 3rd trimesters. Proportions of women achieving defined titers were compared using the χ² test. Results Of 307 women, 190 (62%) were unvaccinated. Fifty and 67 were vaccinated during the 2nd and the 3rd trimesters, respectively. Median enrollment age was 29 years (interquartile range 24–34). Sixteen (5%) women had pre-existing conditions, but none were immunocompromised. GMT ratios comparing vaccinated and unvaccinated women were 5.90 (95% confidence interval [CI] 5.06–6.96) for influenza A/California, 5.39 (95% CI 4.18–6.08) for influenza A/Switzerland, and 5.05 (95% CI 4.43–5.85) for influenza B/Phuket. Similarly, the GMT ratios comparing the 3rd and the 2nd trimester vaccinated women were 2.90 (95% CI 2.54–3.39), 2.82 (95% CI 2.56–3.13), and 2.83 (95% CI 2.56–3.14), respectively. The proportions of women with defined titers for the three vaccine reference viruses did not differ between 2nd and 3rd trimester vaccinated women (titers ≥40: 68–92% versus 70–93%; ≥110: 32% versus 33–63%; and ≥330: 4–10% versus 3–21%). Conclusions Pregnant women vaccinated against influenza had more placental transfer of influenza antibodies to their infants than unvaccinated women. Placental transfer of antibodies was higher among those vaccinated in the 3rd trimester than in the 2nd trimester. There was no difference in the proportions of women achieving antibody titers corresponding to protection against influenza in children. Findings support the current World Health Organization’s recommendation that pregnant women may be vaccinated in either 2nd or 3rd trimester of pregnancy.

The coding-complete genome sequences of 22 chikungunya virus strains collected from the 2018–2019 outbreak in Thailand are reported. All sequences belong to the East/Central/South African (ECSA) genotype and contain two mutations, E1:K211E and E2:V264A, which were previously shown to be associated with increased viral infectivity, dissemination, and transmission in Aedes aegypti .

Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs.

Background: We measured seroconversion to influenza viruses and incidence of symptomatic influenza virus infection in a cohort of children in Bangkok, Thailand. Methods: Children aged ≤6 months were followed for two years for acute respiratory illness (ARI) and had serum specimens taken at 6-month intervals and tested by hemagglutination inhibition (HI) assay. Seroconversion was defined as a >4-fold rise in the HI titers between time points with a titer of >40 in the second specimen. Respiratory swabs were tested by rRT-PCR for influenza. Data were analyzed using generalized linear models. Results: Of 350 children, 266 (76%, 147 were healthy and 119 were high-risk) had ≥2 serum specimens collected before influenza vaccination. During the 2-year follow-up, 266 children contributed 370 person-years of observation, excluding post-vaccination periods. We identified 32 ARI cases with rRT-PCR-confirmed influenza virus infection (7 infections/100 person-years, 95% confidence interval [CI], 4-11). There were 126 episodes of influenza virus infection, resulting in a seroconversion rate of 35 infections/100 person-years (95% CI, 30-42). Rates in healthy and high-risk children did not differ. Conclusions: Influenza virus infection is common during the first two years of life among Thai children. A large proportion of infections may not be detected using the ARI case definition.

Background: HIV-infected persons are at increased risk for severe influenza, yet immune responses to standard dose intramuscular influenza vaccine are suboptimal in this population. Intradermal delivery of influenza vaccine might improve immune response through enhanced stimulation of dendritic cells. Methods: We conducted a randomized, double-blind, controlled trial to compare the immunogenicity of off-label standard dose (15mcg) intradermal (ID) versus standard dose (15mcg) intramuscular (IM) inactive influenza vaccine in HIV-infected men in Bangkok, Thailand. The primary study outcome was seroconversion (minimum titer of 1:40 and ≥4 fold rise in antibody titer) at 1 month post vaccination based on serum hemagglutination inhibition (HI) antibody titers against each vaccine strain. Adverse events in the 7 days following vaccination were also assessed. Results: We enrolled 400 HIV-infected participants; 200 were randomly assigned to receive IM and 200 ID vaccine. Vaccine arms were well-balanced with respect to age, CD4 cell count, HIV viral load and antiretroviral treatment. Percent seroconversion to all (ID 14% vs. IM 15%; p=0.8) or at least one (ID 69% vs. IM 68%; p=0.7) of the three vaccine strains did not differ significantly between ID versus IM vaccine recipients. A higher proportion of participants who received ID vaccine had mild injection site adverse events compared to participants who received IM vaccine (77% versus 27%). Conclusions: There were no significant differences in the immunogenicity of standard dose ID versus IM influenza vaccine in this HIV-infected population in Thailand. Additional strategies to enhance immune responses to influenza vaccine among HIV-infected persons are needed.

Among 368 Thai men who have sex with men with paired serum samples collected before and during the 2009 H1N1 influenza pandemic, we determined influenza A (H1N1)pdm09 seroconversion rates (≥4-fold rise in antibody titers by hemagglutination inhibition or microneutralization assays). Overall, 66 of 232 (28%) participants seroconverted after the first year of A(H1N1)pdm09 activity, and 83 of 234 (35%) participants seroconverted after the second year. Influenza A(H1N1)pdm09 seroconversion did not differ between human immunodeficiency virus (HIV)-infected (55 of 2157 [35%]) and HIV-uninfected (71 of 2211 [34%]) participants (P = .78). Influenza A(H1N1)pdm09 seroconversion occurred in approximately one third of our Thai study population and was similar among HIV-infected and HIV-uninfected participants.