Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1.

The humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63, P = 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = -0.07 to 0.26, P = 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.IMPORTANCE Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.

Rapid Development of gp120-Focused Neutralizing B Cell Responses during Acute Simian Immunodeficiency Virus Infection of African Green Monkeys.

UNLABELLED: The initial phases of acute human immunodeficiency virus type 1 (HIV-1) infection may be critical for development of effective envelope (Env)-specific antibodies capable of impeding the establishment of the latent pool of HIV-1-infected CD4(+) T cells, preventing virus-induced immune hyperactivation to limit disease progression and blocking vertical virus transmission. However, the initial systemic HIV-1 Env-specific antibody response targets gp41 epitopes and fails to control acute-phase viremia. African-origin, natural simian immunodeficiency virus (SIV) hosts do not typically progress to AIDS and rarely postnatally transmit virus to their infants, despite high milk viral loads. Conversely, SIV-infected rhesus macaques (RMs), Asian-origin nonnatural SIV hosts, sustain pathogenic SIV infections and exhibit higher rates of postnatal virus transmission. In this study, of acute SIV infection, we compared the initial systemic Env-specific B cell responses of AGMs and RMs in order to probe potential factors influencing the lack of disease progression observed in AGMs. AGMs developed higher-magnitude plasma gp120-specific IgA and IgG responses than RMs, whereas RMs developed more robust gp140-directed IgG responses. These gp120-focused antibody responses were accompanied by rapid autologous neutralizing responses during acute SIV infection in AGMs compared to RMs. Moreover, acute SIV infection elicited a higher number of circulating Env-specific memory B cells in peripheral blood of AGMs than in the blood of RMs. These findings indicate that AGMs have initial systemic Env-specific B cell responses to SIV infection distinct from those of a nonnatural SIV host, resulting in more functional SIV-specific humoral responses, which may be involved in impairing pathogenic disease progression and minimizing postnatal transmission. IMPORTANCE: Due to the worldwide prevalence of HIV-1 infections, development of a vaccine to prevent infection or limit the viral reservoir remains an important goal. HIV-1-infected humans, as well as SIV-infected nonnatural SIV hosts, develop pathogenic infections and readily transmit the virus to their infants. Conversely, natural SIV hosts do not develop pathogenic infections and rarely transmit the virus to their infants. The immunologic factors contributing to these favorable outcomes in natural SIV hosts could prove invaluable for directing HIV-1 vaccine and therapy design. This study identified distinctions in the specificity and function of the initial systemic SIV envelope-specific B cell response that developed during acute SIV infection in natural and nonnatural SIV host species. Identification of distinct acute B cell responses in natural SIV hosts may inform vaccine strategies seeking to elicit similar responses prior to or during the initial phases of acute HIV-1 infection.

Lack of B cell dysfunction is associated with functional, gp120-dominant antibody responses in breast milk of simian immunodeficiency virus-infected African green monkeys.

The design of an effective vaccine to reduce the incidence of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) via breastfeeding will require identification of protective immune responses that block postnatal virus acquisition. Natural hosts of simian immunodeficiency virus (SIV) sustain nonpathogenic infection and rarely transmit the virus to their infants despite high milk virus RNA loads. This is in contrast to HIV-infected women and SIV-infected rhesus macaques (RhMs), nonnatural hosts which exhibit higher rates of postnatal virus transmission. In this study, we compared the systemic and mucosal B cell responses of lactating, SIV-infected African green monkeys (AGMs), a natural host species, to that of SIV-infected RhMs and HIV-infected women. AGMs did not demonstrate hypergammaglobulinemia or accumulate circulating memory B cells during chronic SIV infection. Moreover, the milk of SIV-infected AGMs contained higher proportions of naive B cells than RhMs. Interestingly, AGMs exhibited robust milk and plasma Env binding antibody responses that were one to two logs higher than those in RhMs and humans and demonstrated autologous neutralizing responses in milk at 1 year postinfection. Furthermore, the plasma and milk Env gp120-binding antibody responses were equivalent to or predominant over Env gp140-binding antibody responses in AGMs, in contrast to that in RhMs and humans. The strong gp120-specific, functional antibody responses in the milk of SIV-infected AGMs may contribute to the rarity of postnatal transmission observed in natural SIV hosts.

Phenotypic and functional profile of HIV-inhibitory CD8 T cells elicited by natural infection and heterologous prime/boost vaccination.

Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8(+) T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8(+) T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8(+) T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8(+) T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8(+) T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1beta (MIP-1beta) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8(+) T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8(+) T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.

The human mitochondrial translation initiation factor 2 gene (MTIF2): transcriptional analysis and identification of a pseudogene.

Mitochondrial translation initiation factor 2 (MTIF2) is nuclear-encoded and functions in mitochondria to initiate the translation of proteins encoded by the mitochondrial genome. To gain insight into mechanisms that regulate MTIF2 gene expression, the genomic copy and the 5' and 3' flanking regions of MTIF2 were isolated using a combination of genomic library screening and polymerase chain reaction (PCR). MTIF2 is approximately 33.5-kb long and contains 16 exons, confirming data from the Human Genome Project. With RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we mapped the transcription start point in human heart tissue to a cytosine residue 296 bp upstream from the translation initiation site. The region surrounding the transcription start point contains consensus binding sites for transcription factors Sp1, nuclear respiratory factor 2 (NRF-2) and estrogen receptor, while enhancer binding sites were identified upstream. Promoter constructs were prepared in a luciferase reporter vector and transiently transfected into 293T cells. The minimal promoter gave an expression level 3.5x higher than the SV40 control (P=0.001), while the construct containing the minimal promoter plus the enhancer region gave a 3.8x higher level of expression compared to the control (P<0.001). We also discovered a pseudogene of MTIF2 and mapped it to chromosome 1p13-12.