Early Treatment with Integrase Inhibitor Achieves a Functional Cure for Pediatric SIV Infection

Neonatal HIV infection causes rapid disease progression to end-stage of AIDS if untreated. To date, there are 29 no reported cases of a genuine functional cure in Pediatric AIDS Clinical Trial Group lacking integrase inhibitor 30 regimen, even when initiated early. In this study, three infant rhesus macaques born on the same day were 31 intravenously infected with SIV on the day of birth and received ART containing tenofovir, FTC, and 32 dolutegravir (DTG) initiated 3 days post infection and continued daily for 9 months. The results showed early 33 ART combination effectively suppressed viral replication to undetectable viremia throughout the treatment 34 course. Remarkably, two of three infants showed complete virologic remission and intestinal CD4+ T cell 35 preservation throughout the period of study after analytical treatment interruption (ATI), as well as undetectable 36 cell-associated proviral DNA in PBMCs, axillary lymph nodes (LNs), and rectal biopsies. However, viral 37 rebound was observed in one of the three infants at two months post ATI. Notably, very low levels of integrated 38 proviral SIV DNA were detected in the lymph node of this animal under cART. Sustained virologic remission 39 was achieved in the other two infants despite absence of protective MHC alleles or neutralizing antibody 40 responses. These findings suggest that early integrase inhibitor-based ART, initiated before irreversible 41 proviral reservoir seeding, can completely suppress viral replication and effectively block new viral genome 42 integration, providing a proof of concept that HIV functional cure and possibly a cure is possible, at least in a 43 proportion of infants postnatally infected with HIV. macaques who were born on the same day and infected with SIV on the day of birth. Our data showed that sustained virologic remission had been achieved throughout the period of study, in two of three postnatally SIV-infected infant rhesus macaques using an early DTG-based ART regimen. Collectively, an optimized early ART regimen, initiated prior to proviral reservoir seeding, can potentially achieve a functional cure in a proportion of HIV-infected infants.

Introduction month 1, albeit SIV RNA was not detected at this time point in this animal, followed by rapid increase of cell-

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associated SIV RNA and proviral DNA once treatment is discontinued (Figs. 2G and 2H), which was 115 consistent with the emergence of viral rebound and SIV positive cells in the lymphoid tissues post ATI (Figs.

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1A, 2A and 2D). Unfortunately, the samples at ART initiation (3 dpi) in these three infants are not available for 117 tracking the presence of proviral DNA. To provide data on proviral reservoir seeding in neonates at 3dpi, we 118 examined cell-associated viral RNA and integrated proviral DNA in archived lymphocytes from blood, spleen 119 and jejunum at this key timepoint in two additional SIV-infected infants. We did not detect proviral DNA in 120 peripheral and lymphoid tissues at least in these two infants by 3dpi, despite cell-associated SIV RNA was still 121 detectable in all tissues examined (Fig. 2I). These findings suggest that small proviral reservoirs, once seeded 122 at an early stage of HIV infection, may be resistant to ART, and that their persistence imposes a high risk of 123 viral rebound when treatment is stopped.

125 Histopathological examination of lymph nodes and rectal biopsies of SIV-infected infant macaques
126 before and after analytical treatment interruption. Given that two of three infants on early ART achieved 127 what appears to be complete viral remission and one did not, potential correlates of protection behind of this 128 finding were explored including potential pathologic changes in LN and rectal tissues, resistant MHC I alleles, 129 anti-Env /neutralizing antibodies, and SIV-specific CD8+ T cells. We previously reported that SIV infection in 130 newborn macaques inhibits follicle and germinal center formation evident by 1-month post infection, resulting in 131 impaired antibody responses 21 . Histopathological analysis of LNs did not demonstrate differences among the 132 experimental infants, but LN tissue in infant 2 at 1 month after early ART, in which lymphoid follicles rarely 133 contained small and reduced germinal centers with low numbers of histiocytes in the medullary sinuses, and 134 the corticomedullary junction was infiltrated by low to moderate numbers of neutrophils (Figs. 3A-3H). There 135 were multifocal aggregates of neutrophils and adjacent crypts contained degenerate neutrophils (crypt 136 abscesses) in rectum in infant 2 post ATI (Fig. 3I). In infants 1 and 3 before and after ATI, the LN cortex was expanded by multifocal paracortical hyperplasia with low numbers of histiocytes in the medullary sinuses, and the jejunum lamina propria was infiltrated by low numbers of lymphocytes and plasma cells (Figs. 3A-3M), 6 which has been frequently observed in animals on daily drug treatment by intramuscular injection.  12,13,14,16,20,23,24,25 . Here we show pediatric 159 virologic remission is achieved in a proportion of postnatally SIV-infected infant macaques on early DTG-based 160 ART.

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Given a unique immune system in developing neonatal infants, viral susceptibility, viral reservoir establishment, 162 and immune responses in neonates exposed to HIV might differ from those in adults 19,20 . All children exposed 163 to HIV before, during, and after birth should receive early antiretroviral (ARV) drugs to reduce the risk of HIV 164 acquisition, morbidity, and mortality 10,15,20,26,27 . Since interventions to prevent mother-to-child transmission integration and proviral reservoir seeding in infants postnatally or perinatally infected with HIV. Another 169 therapeutic option, DTG, is a very well-tolerated, highly effective, and affordable INSTI drug in low-and middle-170 income countries 30 , and achieves more rapid and complete viral suppression compared to dolutegravir 31 .

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Although a study in Botswana shows that DTG used in the early first trimester of pregnancy may be associated 172 with the incidence of neural tube defects (NTDs) in newborns 32 , whereas additional data from this cohort also 173 indicate the risk may only be slightly greater over any non-DTG ART 33

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Cohort Collaboration (EPPICC) study, there is no case reported to achieve a functional cure in the HIV+ infants 184 treated by early ART (immediate treatment to several years of age). Unfortunately, the eventual viral rebound 185 is observed once treatment is discontinued 11,12,13,14,16,20,23,24,25,39 . Our study demonstrates that pediatric HIV 192 proviral DNA at the initiation of early ART, even in blood, has not been fully evaluated or confirmed in these genome integration and subsequent new proviral reservoir seeding in infants. All of these reasons might 197 explain why early ART initiation does not show curative effects in infants perinatally exposed to HIV,

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underlining the need to diagnose tissue proviral reservoirs in infants at the initiation of ART, and to include 199 integrase inhibitors with the cART strategy.

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The

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However, the protective mechanisms of discrepant outcomes in these infant macaques still remain elusive.

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In summary, establishment of small irreversible proviral reservoirs before early ART initiation may probably

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In addition, archived rectal lymphocytes from age-matched SIV naïve infants (n=6, 1-2 years old) were used for 245 the comparative analysis of post ATI rectal CD4+ T cells in the three experimental infants of the current study.

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To quantify integrated proviral DNA, two-step PCR was run in parallel to quantify all viral DNA as previously 274 described 51 . In brief, the pre-amplification reactions were performed using SIV long terminal repeat primer and 63°C for 30 seconds and extension at 72°C for 3 minutes. Next, 2.5 μL of each amplicon was further amplified 280 in triplicate by real-time PCR reaction using 40 cycles at 95°C for 15 seconds and 63°C for 1 minute. Highly

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To identify the SIV RNA in LN and rectal biopsies, formalin-fixed, paraffin-embedded sections (5-μm thickness) 290 were cut and adhered to sialinized glass slides. After deparaffinization in xylene, and dehydration with 50%,

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Reagent-Red (ACD) following the manufacturer's instructions. The slides were denatured at 60°C for 1 h and 293 incubated with pre-warmed (60°C) SIV gag-pol sense probe (ACD) with an additional incubation for 4 h at 294 40°C. The signal was then amplified by Amp1 to Amp6 and identified on tissue sections using a Zeiss digital 295 slide scanner system.

Measurement of SIV gag-specific CD8+ T cells in blood
297 298 SIV gag-specific CD8+ T cells were detected as we previously described 52 . In brief, PBMCs were stimulated 299 by a pool of 15-mer gag peptides (5 μg/ml each peptide), medium only (negative control), or phorbol-12-300 myristate-13-acetate (PMA, 5 ng/ml, Sigma) plus ionomycin (50 μg/ml) (positive control) for 6 h. The cultures 301 also contained brefeldin A (Sigma) and 1 μg/ml anti-CD49d and anti-CD28 co-stimulatory molecules (BD different samples and different cytokine patterns but was typically <0.05% of total CD8+ T cells (median, 309 0.01%). Only samples in which the percentage of cytokine-staining cells was at least twice that of background 310 were considered positive.