I-106 Early treatment initiation in children with vertical HIV infection influences HIV specific immune responses
Authors: Rinaldi Stefano , Cotugno Nicola , Pallikkuth Suresh , Morrochi Elena , Pahwa Rajendra , Palma Paolo , Pahwa Savita 
Published: April 2019

Background:
Despite successes in prevention of mother to child HIV transmission (MTCT), issues related to early diagnosis, ART initiation and adherence play a role in the disease course of HIV infected children. Early ART initiation in HIV infected infants leads to reduced size of HIV reservoirs and curtails clinical progression but role of the immune system in this process is not well understood. With early initiation of ART, HIV infected infants preserve their CD4+ T cells and in some instances remain seronegative for HIV due to limited exposure of B cells to HIV antigen. We are performing immunologic assessments in the HIV Exposed Pediatric Patients (HEPP) Project and discuss a group of children from Rome, Italy. The goal of the present study is to evaluate HIV-specific immunity of vertically HIV infected children in relation to timing of ART initiation.

Methods:
The study group consists of older vertically infected HIV+ children and adolescents who initiated ART at different ages after birth and have maintained durable viral control (plasma HIV-RNA <50 cp/mL). Immunologic studies were performed in 27 children (at ages 2.1–15.7 yrs) of whom 22 had started ART early, at age <6 mo [early treated (ET)] and 5 who started ART later, at age >2 yrs [late treated (LT)] with ET experiencing longer duration of ART (P < 0.05) at the time of study. ET children were also evaluated by HIV serology (Abbott Architect HIV Ag/Ab Combo Assay) to establish if they were seropositive (SP) or seronegative (SN) to HIV. Flow cytometry was performed using BD LSRFortessa for T cells and BD FACS Aria II for B cells. HIV-gag specific CD4+ and CD8+ T cells were investigated by flow cytometry in cryopreserved PBMC for the induction of intracellular cytokines (IL-2, IFN-g, TNF, IL-21) in CD40L+ CD4+ T cells and intracellular cytokines (IL-2, IFN-g, TNF) as well as Perforin, Granzyme B and CD107a in CD69+ CD8+ T cells following 12 hours stimulation with HIV gag PTE peptides. In addition, Env-specific CD4+ T cells were evaluated following 12 hours stimulation of PBMC with Env peptides. IgD+ and IgM+ B cell subsets were determined in gp140+ B cells. Moreover, env-specific and non-specific B cells were sorted to perform transcriptomic analysis with Fluidigm (Biomark). Differences between groups were determined by unpaired or Mann-Whitney t tests if the data were normally distributed or not, respectively.

Results:
In this group of HIV+ children, frequencies of HIV gag-specific CD4+ T cells and CD8+ T cells were similar between ET and LT but differed qualitatively. Gag stimulated CD4+ T cells of ET exhibited higher proportions of IL-21 and IFNg with enrichment of polyfunctional T cells defined by the simultaneous presence of 3 or more cytokines. Gag stimulated CD8+ T cells of ET exhibited higher frequencies of cytotoxic cells (CD107a+Perf+GrzB+).
In ET, serologic evaluation of HIV Ab revealed 6/22 (27.3%) to be SN. The gp140-specific CD4+ T cell frequencies were similar in SN and SP but intracellular IL-21 expression was higher in SP. Frequencies of gp140+ CD19+ B cells were similar between SN and SP, but HIV-specific CD27+ memory B cells were predominantly IgM+ in SN while in SP, the gp140+CD27+ memory B cells were enriched in IgD-cells. Gene expression signatures in gp140+IgM+ cells revealed upregulation of genes involved in both plasma cell differentiation (PRDM1) and GC reaction (BCL6) only in SP after in-vitro stimulation.

Conclusions:
Time of ART initiation after birth has a long-term impact on the quality of the host HIV-specific immune responses in vertically infected HIV+ children with durable virus suppression. Earlier ART initiation is associated with preserved HIV gag-specific CD4+ and CD8+ T cells. Moreover, even in those who become seronegative for HIV, the HIV Env-specific CD4+ T cells and B cells persist, the latter demonstrating a blunted differentiation with enrichment of IgM+ B cells. ET children with consistent viral control are prime candidates for therapeutic approaches aimed at permanent remission of HIV. These studies are supported by ViiV Health Care grant to EPIICAL and NIH grant AI127347 to SP.