BackgroundEarly life microbiota is an important determinant of immune and metabolic development and may have lasting consequences. The maternal gut microbiota during pregnancy or breastfeeding is important for defining infant gut microbiota. We hypothesized that maternal gut microbiota during pregnancy and breastfeeding is a critical determinant of infant immunity. To test this, pregnant BALB/c dams were fed vancomycin for 5 days prior to delivery (gestation; Mg), 14 days postpartum during nursing (Mn), or during gestation and nursing (Mgn), or no vancomycin (Mc). We analyzed adaptive immunity and gut microbiota in dams and pups at various times after delivery.ResultsIn addition to direct alterations to maternal gut microbial composition, pup gut microbiota displayed lower α-diversity and distinct community clusters according to timing of maternal vancomycin. Vancomycin was undetectable in maternal and offspring sera, therefore the observed changes in the microbiota of stomach contents (as a proxy for breastmilk) and pup gut signify an indirect mechanism through which maternal intestinal microbiota influences extra-intestinal and neonatal commensal colonization. These effects on microbiota influenced both maternal and offspring immunity. Maternal immunity was altered, as demonstrated by significantly higher levels of both total IgG and IgM in Mgn and Mn breastmilk when compared to Mc. In pups, lymphocyte numbers in the spleens of Pg and Pn were significantly increased compared to Pc. This increase in cellularity was in part attributable to elevated numbers of both CD4+ T cells and B cells, most notable Follicular B cells.ConclusionOur results indicate that perturbations to maternal gut microbiota dictate neonatal adaptive immunity.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0511-7) contains supplementary material, which is available to authorized users.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings.Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva.Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers.Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

Differences in Bacille Calmette-Guérin (BCG) immunogenicity and efficacy have been reported, but various strains of BCG are administered worldwide. Since BCG immunization may also provide protection against off-target antigens, we sought to identify the impact of different BCG strains on the ontogeny of vaccine-specific and heterologous vaccine immunogenicity in the first 9 months of life, utilizing two African birth cohorts. A total of 270 infants were studied: 84 from Jos, Nigeria (vaccinated with BCG-Bulgaria) and 187 from Cape Town, South Africa (154 vaccinated with BCG-Denmark and 33 with BCG-Russia). Infant whole blood was taken at birth, 7, 15, and 36 weeks and short-term stimulated (12 h) in vitro with BCG, Tetanus and Pertussis antigens. Using multiparameter flow cytometry, CD4+ T cell memory subset polyfunctionality was measured by analyzing permutations of TNF-α, IL-2, and IFN-γ expression at each time point. Data was analyzed using FlowJo, SPICE, R, and COMPASS. We found that infants vaccinated with BCG-Denmark mounted significantly higher frequencies of BCG-stimulated CD4+ T cell responses, peaking at week 7 after immunization, and possessed durable polyfunctional CD4+ T cells that were in a more early differentiated memory stage when compared with either BCG-Bulgaria and BCG-Russia strains. The latter responses had lower polyfunctional scores and tended to accumulate in a CD4+ T cell naïve-like state (CD45RA+CD27+). Notably, BCG-Denmark immunization resulted in higher magnitudes and polyfunctional cytokine responses to heterologous vaccine antigens (Tetanus and Pertussis). Collectively, our data show that BCG strain was the strongest determinant of both BCG-stimulated and heterologous vaccine stimulated T cell magnitude and polyfunctionality. These findings have implications for vaccine policy makers, manufacturers and programs worldwide and also suggest that BCG-Denmark, the first vaccine received in many African infants, has both specific and off-target effects in the first few months of life, which may provide an immune priming benefit to other EPI vaccines.

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Background: The SARS-CoV-2 pandemic has swept the world and poses a significant global threat to lives and livelihoods, with over 16 million confirmed cases and at least 650 000 deaths from COVID-19 in the first 7 months of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect enzyme-linked immunosorbent assay (ELISA) using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n=77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of six weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n=58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and OD values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusions: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

word count: 241 24 Main text word count: 4,667 25 26 Abstract 29crAssphages are a class of bacteriophages that are highly abundant in the human 30 gastrointestinal tract. Accordingly, crAssphage genomes have been identified in most 31human fecal viral metagenome studies. However, we currently have an incomplete 32 understanding of factors impacting the transmission frequencies of these phages 33 between mothers and infants, and the evolutionary pressures associated with such 34 transmissions. Here, we use metagenome sequencing of stool-associated virus-like 35 particles to identify the prevalence of crAssphage across ten South African mother-36 infant dyads that are discordant for HIV infection. We report the identification of a 37 complete 97kb crAssphage genome, parts of which are detected at variable levels 38 across each mother-infant dyad. We observed average nucleotide sequence identities 39 of >99% for crAssphages from related mother-infant pairs but ~97% identities between 40 crAssphages from unrelated mothers and infants: a finding strongly suggestive of 41 vertical mother to infant transmission. We further analyzed patterns of nucleotide 42 diversity across the crAssphage sequences described here, identifying particularly 43 elevated positive selection in RNA polymerase and phage tail protein encoding genes, 44 which we validated against a crAssphage genome from previous studies. Using 16S 45 rRNA gene sequencing, we found that the relative abundances of Bacteroides 46 thetaiotaomicron and Parabacteroides merdae (Order: Bacteroidales) were differentially 47 Importance 53 crAssphages are an ubiquitous member of the human gut microbiome and modulate 54 interactions with key bacterial associates within the order Bacteroidales. However, the 55 role of this interaction in the genomic evolution of crAssphage remains unclear. Across 56 a longitudinally sampled cohort of ten South African mother-infant dyads, we use 57 metagenome sequencing of the fecal virome and 16S rRNA gene sequencing of the 58 fecal bacterial microbiota to elucidate the ecological and evolutionary dynamics of these 59interactions. Here, we demonstrate elevated levels of crAssphage average nucleotide 60 identity between related mother-infant dyads as compared to unrelated individuals, 61suggesting vertical transmission. We report strong positive selection in crAssphage 62 RNA polymerase and phage tail protein genes. Finally, we demonstrate that 63 crAssphage abundance is linearly correlated (P < 0.014) with the abundance of two 64 bacterial taxa, Bacteroides thetaiotaomicron and Parabacteroides merdae. These 65 results suggest that phage-bacterial interactions may help shape ecological and 66 evolutionary dynamics in the gut. 67 68

Table of contents A1 Introduction to the 2nd synchronicity forum of GHRI/CHVI-funded Canadian and African HIV prevention and vaccine teams O1 Voluntary medical male circumcision for prevention of heterosexual transmission of HIV in adult males in Soweto: What do indicators and incidence rate show? Hillary Mukudu, Neil Martinson, Benn Sartorius O2 Developing a peer-led community mobilization program for sex workers in Soweto: HIV risk and demographics Jenny Coetzee, Janan Dietrich, Kgaugelo Mokgatswana, Rachel Jewkes, Glenda E. Gray O3 Salient beliefs about adherence: A qualitative survey conducted as part of the demonstration study on "treatment as prevention" (TasP) and "pre-exposure prophylaxis" (PrEP) among female sex workers (FSWS) in Cotonou, Benin Marylène Dugas, Luc Béhanzin, Fernand A. Guédou, Marie-Pierre Gagnon, Michel Alary O4 Relative perception of risk as a driver of unsafe sexual practices among key populations: Cases of fisherfolk and women and their partners involved in multiple sexual partnerships in Uganda Rwamahe Rutakumwa, Martin Mbonye, Thadeus Kiwanuka, Sarah Nakamanya, Richard Muhumuza, Winfred Nalukenge, Janet Seeley O5 Exploring the acceptability of new biomedical HIV prevention technologies among MSM, adolescents and heterosexual adults in South Africa Millicent Atujuna, Melissa Wallace, Ben Brown, Linda Gail Bekker, Peter A. Newman O6 HIV-susceptible target cells in foreskins after voluntary medical male circumcision in South Africa Rushil Harryparsad, Abraham J. Olivier, Heather B. Jaspan, Douglas Wilson, Janan Dietrich, Neil Martinson, Hillary Mukudu, Nonhlanhla Mkhize, Lynn Morris, Gianguido Cianci, Minh Dinh, Thomas Hope, Jo-Ann S. Passmore, Clive M. Gray O7 HIV-1 proteins activate innate immune responses via TLR2 heterodimers Bethany M. Henrick, Xiao-Dan Yao, Kenneth L. Rosenthal, the INFANT Study Team O8 Characterization of an innate factor in human milk and mechanisms of action against HIV-1 Bethany M. Henrick, Xiao-Dan Yao, Anna G. Drannik, Alash’le Abimiku, Kenneth L. Rosenthal, the INFANT Study Team O9 Secretor status and susceptibility to HIV infections among female sex workers in Nairobi, Kenya Nadia Chanzu, Walter Mwanda, Julius Oyugi, Omu Anzala O10 Natural Killer cell recall responsiveness to Gag-HIV-1 peptides of HIV-1 exposed but uninfected subjects are associated with peripheral CXCR6+ NK cell subsets Moustapha Mbow, Sabelle Jallow, Moussa Thiam, Alberta Davis, Assane Diouf, Cheikh T. Ndour, Moussa Seydi, Tandakha N. Dieye, Souleymane Mboup, Martin Goodier, Eleanor Rilley, Assan Jaye O11 Profiles of resistance: Local innate mucosal immunity to HIV-1 in commercial sex workers Xiao-Dan Yao, RW. Omange, Bethany M. Henrick, Richard T. Lester, Joshua Kimani, T. Blake Ball, Francis A. Plummer, Kenneth L. Rosenthal ...

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Mechanisms such as defects in the Th1 response to Mycobacterium tuberculosis in HIV-infected persons have been widely reported. However, Th1-independent mechanisms also contribute to protection against TB. To identify a broader spectrum of defects in TB immunity during HIV infection, we examined IL-17A and IL-22 production in response to mycobacterial Ags in peripheral blood of persons with latent TB infection and HIV coinfection. Upon stimulating with mycobacterial Ags, we observed a distinct CD4 + Th lineage producing IL-22 in the absence of IL-17A and IFN-g. Mycobacteria-specific Th22 cells were present at high frequencies in blood and contributed up to 50% to the CD4 + T cell response to mycobacteria, comparable in magnitude to the IFN-g Th1 response (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed that their memory differentiation was similar to M. tuberculosisspecific Th1 cells (i.e., predominantly early differentiated CD45RO + CD27 + phenotype). Moreover, CCR6 and CXCR3 expression profiles of Th22 cells were similar to Th17 cells, whereas their CCR4 and CCR10 expression patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, mycobacterial IL-22 responses were 3-fold lower in HIV-infected persons compared with uninfected persons, and the magnitude of responses correlated inversely with HIV viral load. These data provide important insights into mycobacteria-specific Th subsets in humans and suggest a potential role for IL-22 in protection against TB during HIV infection. Further studies are needed to fully elucidate the role of IL-22 in protective TB immunity.