Nonsecretor Histo-blood Group Antigen Phenotype Is Associated With Reduced Risk of Clinical Rotavirus Vaccine Failure in Malawian Infants

Background Histo–blood group antigen (HBGA) Lewis/secretor phenotypes predict genotype-specific susceptibility to rotavirus gastroenteritis (RVGE). We tested the hypothesis that nonsecretor/Lewis-negative phenotype leads to reduced vaccine take and lower clinical protection following vaccination with G1P[8] rotavirus vaccine (RV1) in Malawian infants Methods A cohort study recruited infants receiving RV1 at age 6 and 10 weeks. HBGA phenotype was determined by salivary enzyme-linked immunosorbent assay (ELISA). RV1 vaccine virus shedding was detected by quantitative real-time polymerase chain reaction (qRT-PCR) in stool collected on alternate days for 10 days post-immunization. Plasma rotavirus–specific immunoglobulin A was determined by ELISA pre- and post-immunization. In a case-control study, HBGA phenotype distribution was compared between RV1-vaccinated infants with RVGE and 1:1 age-matched community controls. Rotavirus genotype was determined by RT-PCR. Results In 202 cohort participants, neither overall vaccine virus fecal shedding nor seroconversion differed by HBGA phenotype. In 238 case-control infants, nonsecretor phenotype was less common in infants with clinical vaccine failure (odds ratio [OR], 0.39; 95% confidence interval [CI], 0.20–0.75). Nonsecretor phenotype was less common in infants with P[8] RVGE (OR, 0.12; 95% CI, 0.03–0.50) and P[4] RVGE (OR, 0.17; 95% CI, 0.04–0.75). Lewis-negative phenotype was more common in infants with P[6] RVGE (OR, 3.2; 95% CI, 1.4–7.2). Conclusions Nonsecretor phenotype was associated with reduced risk of rotavirus vaccine failure. There was no significant association between HBGA phenotype and vaccine take. These data refute the hypothesis that high prevalence of nonsecretor/Lewis-negative phenotypes contributes to lower rotavirus vaccine effectiveness in Malawi

Emergence of Double- and Triple-Gene Reassortant G1P[8]Rotaviruses Possessing a DS-1-Like Backbone after RotavirusVaccine Introduction in Malawi

To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunisation programmes. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix™) into its immunisation schedule in 2012. Utilising a surveillance platform of hospitalised rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 - 2012) and after (2013 - 2014) vaccine introduction. Analysis of whole genomes obtained through next generation sequencing revealed that all randomly-selected pre-vaccine G1P[8] strains sequenced (n=32) possessed a Wa-like genetic constellation, whereas post-vaccine G1P[8] strains (n=18) had a DS-1-like constellation. Phylodynamic analyses indicated that post-vaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990's, hence classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981-1994; their evolutionary rates ranged from 9.7 x 10(-4)-4.1 x 10(-3) nucleotide/substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.ImportanceThe error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine-escape mutants. While multiple African countries have introduced rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the post-vaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998-2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting limited effect in recognition of G1 specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation

Community transmission of rotavirus infection in a vaccinated population in Blantyre, Malawi: a prospective household cohort study.

BACKGROUND: Rotavirus vaccine effectiveness is reduced among children in low-income countries. Indirect (transmission-mediated) effects of rotavirus vaccine might contribute to the total population effect of vaccination. We aimed to examine risk factors for transmission of rotavirus to household contacts in Blantyre, Malawi, and estimated the effectiveness of rotavirus vaccine in preventing transmission of infection to household contacts. METHODS: In this prospective household cohort study, we recruited children born after Sept 17, 2012, and aged at least 6 weeks (vaccine-eligible children) with acute rotavirus gastroenteritis and their household contacts, in four government health facilities in Blantyre, Malawi. Clinical data, a bulk stool sample, and 1-2 mL of serum were collected from case children at presentation. Clinical data and stool samples were also prospectively collected from household contacts over 14 days from presentation. A single stool sample was collected from control households containing asymptomatic children who were frequency age-matched to case children. Samples were tested for rotavirus using semi-quantitative real-time PCR and for anti-rotavirus IgA using a semi-quantitative sandwich ELISA. Risk factors for household transmission of rotavirus infection and clinical disease, including disease severity and faecal shedding density, were identified using mixed effects logistic regression. Vaccine effectiveness against transmission was estimated as 1 minus the ratio of secondary attack rates in vaccinated and counterfactual unvaccinated populations, using vaccine effectiveness estimates from the associated diarrhoeal surveillance platform to estimate the counterfactual secondary attack rate without vaccination. FINDINGS: Between Feb 16, 2015, and Nov 11, 2016, we recruited 196 case households (705 members) and 55 control households (153 members). Household secondary attack rate for rotavirus infection was high (434 [65%] of 665 individuals) and secondary attack rate for clinical disease was much lower (37 [5%] of 698). Asymptomatic infection in control households was common (40 [28%] of 144). Increasing disease severity in an index child (as measured by Vesikari score) was associated with increased risk of transmission of infection (odds ratio 1·17 [95% CI 1·06-1·30) and disease (1·28 [1·08-1·52]) to household contacts. Estimated vaccine effectiveness against transmission was 39% (95% CI 16-57). INTERPRETATION: Rotavirus vaccine has the potential to substantially reduce household rotavirus transmission. This finding should be considered in clinical and health economic assessments of vaccine effectiveness. FUNDING: Wellcome Trust, US National Institutes of Health, and US National Institute of Allergy and Infectious Diseases

AstraZeneca COVID-19 vaccine induces robust broadly cross-reactive antibody responses in Malawian adults previously infected with SARS-CoV-2

Background: Binding and neutralising anti-Spike antibodies play a key role in immune defence against SARS-CoV-2 infection. Since it is known that antibodies wane with time and new immune-evasive variants are emerging, we aimed to assess the dynamics of anti-Spike antibodies in an African adult population with prior SARS-CoV-2 infection and to determine the effect of subsequent COVID-19 vaccination. Methods: Using a prospective cohort design, we recruited adults with prior laboratory-confirmed mild/moderate COVID-19 in Blantyre, Malawi, and followed them up for 270 days (n = 52). A subset of whom subsequently received a single dose of the AstraZeneca COVID-19 vaccine (ChAdOx nCov-19) (n = 12). We measured the serum concentrations of anti-Spike and receptor-binding domain (RBD) IgG antibodies using a Luminex-based assay. Anti-RBD antibody cross-reactivity across SARS-CoV-2 variants of concern (VOC) was measured using a haemagglutination test. A pseudovirus neutralisation assay was used to measure neutralisation titres across VOCs. Ordinary or repeated measures one-way ANOVA was used to compare log10 transformed data, with p value adjusted for multiple comparison using Šídák's or Holm-Šídák's test. Results: We show that neutralising antibodies wane within 6 months post mild/moderate SARS-CoV-2 infection (30–60 days vs. 210–270 days; Log ID50 6.8 vs. 5.3, p = 0.0093). High levels of binding anti-Spike or anti-RBD antibodies in convalescent serum were associated with potent neutralisation activity against the homologous infecting strain (p < 0.0001). A single dose of the AstraZeneca COVID-19 vaccine following mild/moderate SARS-CoV-2 infection induced a 2 to 3-fold increase in anti-Spike and -RBD IgG levels 30 days post-vaccination (both, p < 0.0001). The anti-RBD IgG antibodies from these vaccinated individuals were broadly cross-reactive against multiple VOCs and had neutralisation potency against original D614G, beta, and delta variants. Conclusions: These findings show that the AstraZeneca COVID-19 vaccine is an effective booster for waning cross-variant antibody immunity after initial priming with SARS-CoV-2 infection. The potency of hybrid immunity and its potential to maximise the benefits of COVID-19 vaccines needs to be taken into consideration when formulating vaccination policies in sub-Saharan Africa, where there is still limited access to vaccine doses

Emergence of double- and triple-gene reassortant G1P[8] rotaviruses possessing a DS-1-like backbone after rotavirus vaccine introduction in Malawi

To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains (n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10−4 to 4.1 × 10−3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation. IMPORTANCE: The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine escape mutants. While multiple African countries have introduced a rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the postvaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998 to 2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting a limited effect on the recognition of G1-specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation

Etiology of Diarrhea Among Hospitalized Children in Blantyre, Malawi, Following Rotavirus Vaccine Introduction: A Case-Control Study.

Despite rotavirus vaccination, diarrhea remains a leading cause of child mortality. We collected stool specimens from 684 children <5 years of age hospitalized with diarrhea (cases) and 527 asymptomatic community controls for 4 years after rotavirus vaccine introduction in Malawi. Specimens were tested for 29 pathogens, using polymerase chain reaction analysis. Three or more pathogens were detected in 71% of cases and 48% of controls. Pathogens significantly associated with diarrhea included rotavirus (in 34.7% of cases and 1.5% of controls), enteric adenovirus (in 29.1% and 2.7%, respectively), Cryptosporidium (in 27.8% and 8.2%, respectively), heat-stable enterotoxin-producing Escherichia coli (in 21.2% and 8.5%, respectively), typical enteropathogenic E. coli (in 18.0% and 8.3%, respectively), and Shigella/enteroinvasive E. coli (in 15.8% and 5.7%, respectively). Additional interventions are required to prevent diarrhea due to rotavirus and other common causal pathogens

Direct and possible indirect effects of vaccination on rotavirus hospitalisations among children in Malawi four years after programmatic introduction.

IntroductionDespite increased use of vaccine in routine immunisation, rotavirus remains a major cause of acute gastroenteritis (AGE) in low-income countries. We describe rotavirus prevalence and hospitalisation in Malawi pre and four years post vaccine introduction; provide updated vaccine effectiveness (VE) estimates; and assess rotavirus vaccine indirect effects.MethodsChildren under five years of age presenting to a referral hospital in Blantyre with AGE were recruited. Stool samples were tested for rotavirus using Enzyme Immunoassay. The change in rotavirus prevalence was evaluated using Poisson regression. Time series analysis was used to further investigate trends in prevalence over time. VE against rotavirus diarrhoea of any severity was estimated using logistic regression. Indirect effects were estimated by evaluating rotavirus prevalence in unvaccinated children over time, and by comparing observed reductions in incidence of rotavirus hospitalisation to those expected based on vaccine coverage and trial efficacy estimates.Results2320 children were included. Prevalence of rotavirus in hospitalised infants (ConclusionsFollowing rotavirus vaccine introduction in Malawi, prevalence of rotavirus in hospitalised children with AGE has declined significantly, with some evidence of an indirect effect in infants. Despite this, rotavirus remains an important cause of severe diarrhoea in Malawian children, particularly in the second year of life

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing

Background: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7 % of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8 % of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pi