HIV disease progression is associated with phenotypic and functional abnormalities of B cells, several of which are driven by the chronic immune activation associated with HIV replication. However, few studies have evaluated the role of the humoral immune response in spontaneous control of viral replication. In adults, the median frequency of HIV-specific memory B cells in ECs was significantly higher compared to that found in individuals receiving ART (57). While this has not been evaluated in pediatric ECs, a recent study has highlighted B cell dysfunction as a hallmark of HIV disease progression in children with HIV. In this study, a cohort of pediatric long-term non-progressors (LTNPs; n=20) had lower naïve and resting memory B cell frequencies compared to uninfected children (58), while tissue-like memory B cells were lower in LTNPs compared to pediatric progressors. Interestingly, plasma levels of B lymphocyte stimulator (BLyS), a known regulator of B cell function and survival, were lower in pediatric LTNPs compared to progressors. In adults, BLyS expression levels are unaltered, and less B cell dysregulation was observed in non-progressors (59).

Recent work has highlighted a potential role for non-neutralizing HIV-specific antibodies in protection from disease progression and viral control, implicating features that could be exploited in attempts to achieve HIV cure. Muenchhoff et al. described that a subset of ART-naïve children with HIV who maintained normal CD4+ T cells also known as pediatric non progressors (PNPs) had higher p24-specific IgG levels than children under ART, and these anti-p24 IgG were mostly of the IgG1 subclass (60). Meanwhile in PPs, there was a greater contribution to IgG3 subclass to the p24-specific IgG responses. IgG1 mediates antibody responses to viral infection while IgG3 regulates proinflammatory effector functions (61). This suggests that in PNPs viral replication is under control without chronic inflammation, while PPs have more inflammatory responses. Interestingly, when compared to uninfected children, PNPs had no changes in bulk IgG galactosylation, which corresponds to low immune activation and inflammation (60). In PPs, agalactosylated IgG glycans were expanded, while digalactosylated glycans were decreased compared to HIV-uninfected children. Moreover, PNPs demonstrated increased HIV-specific Fc-mediated IgG effector functions, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), compared to progressors (60). Interestingly, robust ADCC (62, 63) and overall superior polyfunctional Fc dependent activity (64, 65) have been consistently observed in adult ECs. Broadly neutralizing antibodies (bNAbs) arise early with higher breadth and potency in children than in adults with HIV (66–68). While bNAbs in children mostly target similar epitopes to adult bNAbs (69), they also showed remarkable polyclonalities with some bNAbs targeting up to 4 distinct epitopes (69, 70). Altogether, these data suggest that successful monoclonal antibody-based therapeutics for viral remission in pediatric HIV will likely need to exploit neutralizing and non-neutralizing properties of HIV-specific bNAbs to control and mediate clearance of infected cells.

There are several opportunities in the early life immune system that could be exploited towards an HIV cure. The immunotolerant and naive immune environment can allow low levels of activation and immune system maturation is highly adaptable. A better understanding of the early life immune ontogeny and mechanism of viral control in pediatric ECs could provide insight into novel immune-based strategies towards clearance of latently infected cells and viral remission in children with HIV.

The latent HIV reservoirs are heterogenous in both cell types and tissue localizations within the body. Various CD4+ T cell types are susceptible to HIV infection including naïve cells, long-lived memory cells (stem cell and central memory cells), and terminally differentiated cells (90), but central memory cells CD4+ T cells are the most vital to the maintenance of the latent reservoir (91). HIV-infected CD4+ T cells exist broadly throughout the body and have been found in the gut-associated lymphoid tissue, genital tract, and lymph nodes (92). In addition to CD4+ T cells, myeloid immune cells have been implicated as HIV reservoirs. Macrophages are known to be infected by HIV (91, 93) and in vitro infectivity studies showed that they are not killed by HIV, suggesting that they are likely HIV reservoirs (93). Tissue resident macrophages are highly specialized cells and varied in susceptibility to HIV infection. Alveolar macrophages and microglial cells are susceptible to HIV infection and contribute to the formation of cell reservoirs in the lung and brain, respectively (94, 95).

The majority of studies that focused on kinetics and origin of persistent latent reservoirs that eventually contribute to viral rebound when ART is interrupted were conducted in adults with HIV or adult NHPs. Infant NHPs are highly relevant and translatable models for the study of pediatric HIV infection and persistent latent reservoirs. Several studies using oral infant NHP Simian-Human Immunodeficiency Virus (SHIV) infection model that mimic breastmilk transmission of HIV, the gastrointestinal (GI) tract was identified as major source of viral RNA during ART and upon viral rebound (96–98). Following ART discontinuation, while the GI tract showed the most rapid increase in viral RNA, virus was also detected in the nasal-associated lymphoid tissue (NALT), axillary lymph node, and spleen (97). An earlier study described that while the oral mucosa and tonsils are generally the anatomical location for initial establishment of a small founder population of infected cells, the anatomical location of persistent latent reservoirs in SHIV-infected infant NHPs continues to spread into distal lymph nodes and other organs including the brain, lungs, intestines, pancreas, and spleen (99).

In addition, due to differences in age-related immune landscapes, the latent HIV reservoirs in adults and children are defined by distinct characteristics. In perinatally-acquired HIV in children, the latent reservoir consists of transitional memory CD4+ T cells as opposed to the mostly central memory CD4+ T cells observed in adults (100, 101). Additionally, perinatally-infected children have the unique opportunity of initiating ART early, which has been reported to limit the establishment of the initial latent HIV reservoir (12, 102, 103). A recent study demonstrated that reactivation of latent HIV reservoirs with PMA/ionomycin from adolescent who were perinatally-infected and treated within the first 24-months of life, resulted in similar rate of cell activation but slower and lower magnitude of proviral load when compared to infected adults (104), suggesting that latent viral reservoirs in children were more resistant to ex vivo reactivation. This study highlights differences in inducibility of latent viral reservoirs resulting from perinatal versus adult HIV infections, which could impact strategies toward latent reservoirs clearance and viral remission in children.

CD4+ T cell immune signatures associated with the size of viral reservoir in adolescents and children who were vertically infected with HIV were recently described (105). Rinaldi et al., reported that while co-expression of PD-1 and TIGIT was associated with CD4+ T cell viral reservoir in both children and adult, the frequencies of PD-1 and TIGIT expressing CD4+ T cells along with HIV-specific CD4 T cells were able to discriminate between lower and higher viral reservoirs in perinatally-infected children (105). This finding suggests that while some immune signatures that could be targeted by interventions or therapies towards clearance of latent viral reservoirs are similar in children and adults, there are also unique signatures that immune-based strategies aimed at permanent HIV remission in children could target.

The goal of therapeutic vaccination is to improve the functional capacity of the host immune system to kill infected CD4+ T cells and/or neutralize circulating viruses (109). Several therapeutic HIV vaccines have been assessed preclinically and clinically for their safety, immunogenicity, and effectiveness but these studies have largely focused on adult HIV population or SIV/adult nonhuman primate (NHP) models (107, 109). It is well established that perinatally-infected infants and children who are treated early with ART tend to have a small pool of latent viral reservoir, normal development of B and T cell compartments, and a high capacity to regenerate an immune repertoire (110, 111). While lessons from the “Mississippi baby” have indicated that early ART treatment is not enough to achieve life-long HIV remission, such characteristics do render perinatally-infected children as an ideal group to test therapeutic vaccines for functional HIV cure (110, 112). However, to date, only two clinical and one preclinical NHP therapeutic HIV/SIV vaccine studies have been conducted with a focus of HIV cure in infants and children ( Table 1 ).

The Pediatric AIDS Clinical Trials Group 218 (ACTG 218 or NCT00000762) was the first clinical study conducted to evaluate the safety and immunogenicity of viral envelope proteins as therapeutic vaccine in infants and children with HIV. The rationale behind this study was that immunization with HIV envelope (Env) protein-based vaccines could alter the landscape of naïve immune responses to HIV and thereby delaying viral rebound. Three HIV Env-based vaccines were administered to infants and children with HIV aged 1 month to 18 years at the time of entry and then at 1, 2, 3, 4 and 6 months later. The vaccines were well tolerated with no adverse effects, secondary infections, or change in viral replication status. Around 65% of vaccinees demonstrated moderate to strong antigen-specific antibody responses and up to 56% of vaccinees exhibited vaccine antigen-specific lymphoproliferative responses, indicative of immunogenicity of the therapeutic vaccine (113).

The First Pediatric Randomized Trial (PEDVAC or NCT04301154) evaluated safety and immunogenicity of the multiclade, multigene HIV DNA therapeutic vaccine (HIVIS) in 10 HIV infected and ART treated children of 6 to 16 years of age. Administration of an HIV DNA vaccine construct expressing HIV-1 subtypes A, B, and C, Env, Rev, Gag, and reverse transcriptase (RT) was well tolerated and exhibited a safe profile except for local irritation, erythema with or without swelling or usual itching at the site of injection. Unlike, non-vaccinated children, vaccinees exhibited increased percentage of HIV-specific perforin-producing CD8+ T cells, compared to baseline, highlighting the immunogenicity of therapeutic HIV-DNA vaccines (114–116). However, there was no evidence that the vaccine had an impact on latent viral reservoir and viral rebound.

Preclinical studies demonstrated the promise of therapeutic vaccination with Ad26/MVA (recombinant adenovirus serotype 26 (Ad26) prime, modified vaccinia Ankara (MVA) boost) encoding SIV gag, pol, env in combination with a toll-like receptor 7 (TLR-7) agonist as an adjuvant against Simian Immunodeficiency Virus (SIV) using an adult NHP model (117). This vaccine regimen was recently translated to an infant SIV NHP model that simulated postnatal HIV infection through breastfeeding with ART-mediated suppression of viremia (118). In this study, a total of 8 infant NHPs were orally infected with SIVmac251 and treated with ART at 4 weeks post-infection followed by multiple immunization with Ad48/MVA encoding SIV gag, pol env plus TLR7 while remining on ART. The vaccinated group showed greater magnitude and breadth of polyfunctional CD4+ and CD8+ T cells when compared to control group and, as anticipated, the administration of the TLR-7 agonist (GS-986) led to activation of monocytes and T cells. This finding was similar to the observations found in adult NHPs (117). Additionally, the levels of SIV gp120-specific antibodies were significantly increased following the MVA boost. However, despite these robust vaccine-induced cellular and humoral immune responses, the size of viral reservoirs, time to viral rebound, and viral set point following analytical ART interruption (ATI) were similar in the vaccine and control groups. These findings suggest that while therapeutic vaccine-induced immunity may induce antiviral T and B cell responses, these responses may be insufficient to modulate viral rebound dynamics during ATI. Thus, there is a need to identify T and B cell responses that could potentially impact the latently infected HIV reservoir and to combine therapeutic vaccines with other immune therapies such as potent latency reversing agents (LRAs), passive transfer of broadly neutralizing antibodies (bNAbs), and check point blockade if this approach is pursued as strategy for HIV remission in children.

Recent development in the use of antibodies for HIV therapy showed promise in the ability of antibody-based strategies to reduce plasma viremia and to mediate clearance of infected cells to further delay viral rebound following ATI (119–123). Emerging evidence supports the effect of antibody-based therapies on persistent latent HIV reservoirs, which could potentially induce viral remission when used alone or in combination with ART and other novel therapeutic modalities (121).

Passive infusion with highly potent broadly neutralizing antibodies (bNAbs) against distinct HIV Env regions may represent an advantageous strategy due to the ability of bNAbs to neutralize free virions via the Fab domain and to engage cognate receptors on host innate and adaptive via the Fc domain. Passive infusion with single or combination bNAbs is actively evaluated as therapeutic strategy for HIV remission in the clinics by measuring the effects of antiviral activities of bNAbs on plasma viremia and viral suppression following ATI as well as impact on viral reservoir by delaying viral rebound (121). Most of these studies were conducted in adults with acute or chronic infections while on ART and similar studies in pediatric HIV population are lacking. To date, only two ongoing clinical trials of bNAb therapy as part of an HIV treatment strategy have been conducted in infants and children ( Table 2 ). Passive infusion of the unmodified version and the long acting (LS) version of the CD4 binding site (CD4bs), VRC01, was shown to be safe and well tolerated in HIV-exposed infants (124, 125). Similarly, passive infusion of the modified and extended half-life version of the VRC01 with the LS mutation in combination with the V3-glycan bNAb, 10-1074, was also safe and well tolerated in the pediatric population (125). The ability of monotherapy and dual therapy using bNAbs to maintain viral suppression and delay viral rebound following ATI in the pediatric HIV population is still under investigation.

Preclinical data from SHIV infection in adult NHP models demonstrated that administration of single or combination bNAb therapy could potentially target, control, and eliminate viral reservoir, suggesting that ART free remission is attainable following ATI (126–132). These mono- or dual-bNAb therapies were administered either alone or in combination with other interventions strategies, including therapeutic vaccines (126, 127) and immune modulatory agents (130–133). Depletion of CD8+ T cells resulted in transient increase of plasma viremia in animals with controlled viral suppression, suggesting that mechanism of viral control is likely co-dependent on CD8+ T cell immune responses (126, 127). The potential immunomodulatory effect of bNAb therapy on CD8+ T cells was recently demonstrated in adults in the clinic, in which administration of combination bNAb therapy (3BNC117 and 10-1074) was associated with an increase in Gag-specific CD8+ T cells in the blood (134). However, whether the increase in HIV-specific CD8+ T cell immune responses contribute to viral control and suppression in the absence of ART remains to be seen. Taken together, these studies highlight the potential of bNAb therapy as an alternative or adjuvant to ART in mediating viral control and/or augmenting the host endogenous immune responses for viral control. Given the unique characteristics of early life immunity as discussed above, it is imperative that effort to advance the use of bNAb therapy in pediatric HIV be tailored to the developing immune system cell composition, activation state, and milieu. Indeed, the highly tolerogenic and anti-inflammatory immune environment during early life as well as differences in pathogenesis between adults and children with HIV may influence efficacy of bNAb therapy towards viral control and remission the pediatric HIV population (135). Thus, the endogenous immune responses in pediatric HIV could potentially be harnessed towards viral elimination and clearance of persistent reservoir to achieve viral remission in the absence of ART. Future studies should aim to investigate the effect of bNAb therapy alone or in combination with therapeutic vaccines and other immunomodulatory agents in augmenting endogenous immune responses as a potential strategy for HIV cure in the in the context of early life immunity.

Numerous efforts are currently being developed to ensure consistent high levels of bNAbs in the circulation including various modifications to extend their half-lives (120, 136–139) and expression of bNAbs using viral vectors (122, 140, 141). Additional strategies are currently being developed to enhance the ability of Fc effector functions, including antibody-mediated cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), to clear HIV-infected cells (121, 122, 142–144). The Fc region enhancement can be accomplished by (i) altering the antibody isotype (142, 143), (ii) modifying the antibody Fc domain amino acid sequences (139, 144), and (iii) modifying the antibody Fc glycans (122, 145). These approaches modulate antiviral breadth and potency of bNAb therapy by increasing bNAbs affinity to cognate Fc receptors on host immune cells and host complement proteins for elimination of HIV-infected cells. These current novel modalities represent tremendous opportunities for pediatric HIV cure. Passively acquired and endogenous ADCC-mediating antibodies contributed to better disease outcome in infants with HIV (146–149). This suggests that passive immunization with bNAbs exhibiting enhanced antiviral functions could be especially attractive for pediatric HIV treatment.

One major challenge in bNAb therapy is the emergence of viral escape to one or more bNAbs due to selection or mutation once the plasma levels of passively administered bNAbs start to decline (150). Antibody engineering through generation of bi- (151, 152) and tri-specific (153–155) bNAbs against HIV have been demonstrated to increase potency and breadth of combination bNAb therapy. An attractive option to improve bNAb performance and overcome their intrinsic limitations is the development of antibody-like molecules, in which antibodies are engineered to (i) improve affinity and recognition of viral epitopes, (ii) combine bi- or tri-specificities, and/or (iii) modify Fc domains to improve half-life and increase engagement of effector cells (156). Overall, there are two major classes of bi- or tri-specific antibody-like molecules: those with or without an antibody Fc region (157).

Asokan and colleagues constructed four different bi-specific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region: VRC07×10E8, VRC07×PGT121, VRC07×PG9-16, and 10E8×PG9-16. All four bi-specific IgGs neutralized over 94% of antigenically diverse viruses in a panel of 206 HIV-1 strains, with VRC07xPG9-16 showing the most positive neutralization profile (158). In a step further in the development of these molecules, a tri-specific antibody-like molecule was generated to include bNAbs with 3 HIV Env specificities, N6 (targeting the CD4bs), PGDM1400 (targeting the V2 glycan), and 10E8v4 (targeting the MPER). This tri-specific antibody-like molecule neutralized clade A SHIV BG505 more potently than any of the parental bNAbs (154), showing comparable potency but greater breadth than a previously developed tri-specific molecule including VRC01/PGDM1400/10E8v4 specificities (153). The newly improved tri-specific antibody-like molecule (N6-CD4bs/PGDM1400-V2 glycan/10E8v4-MPER) was shown to reduce viremia from 100- to 1000-fold in viremic SHIV-infected adult NHPs (154) although transient viral rebound was observed when ART was interrupted, likely due to decline of antibody-like molecule level. However, similar to prior observations with mono- and dual-bNAb therapy, rebound viremia was returned to low levels via CD8+ T cell mediated viral control (154).

Another interesting approach to engineer antibody-like molecule against HIV involves combining a domain to target the virus protein and another domain to target host cell receptors. Mainly, two different constructs have been developed following this strategy: Bi-specific T/NK cell engagers (BiTEs/BiKEs) and Dual Affinity Re-targeting Molecules (DARTs). BiTEs are composed of antibody domain targeting the HIV-1 Env protein gp120 through B12 or VRC01 and an anti-human CD3 single chain variable fragment (scFv) was shown to redirect lysis of HIV gp120-transfected CHO cells in vitro, as well as inhibited HIV replication in both HIV-infected PBMCs and macrophages cocultured with autologous CD8+T cells (159). Similarly, BiKEs are composed of antibody domains D6 and E11, which bind to human CD16 present on NK cells, and a soluble CD4 domain, which binds to HIV gp120. BiKEs activate NK cells in the presence of target cells in and mediate infected target cell lysis (160). DARTs are novel bi-specific-based constructs that seem to offer improved stability, manufacturability, and potency compared to the BiTEs (161). The variable domains of the two antigen-binding moieties in DART molecules are incorporated into a disulfide-linked heterodimer, stabilizing the structure. DART molecules with HIV-1 Env and CD3 specificities were evaluated for their capacity to recruit and redirect cytotoxic T effector cells to primary HIV infected CD4+ T cells.

Unfortunately, all studies with bi/tri-specific molecules so far focused on cells from adult participants, leaving unknown the potential as therapy for pediatric infection. Importantly, immune system development during early life will probably affect the therapeutic potential of bispecific molecules, and thus children’s effector cells might have improved activity profiles.

Latency reversing agents (LRAs) are small-chemical compounds employed to activate and expose latently HIV-infected cells to the host immune system (92). LRAs target different mechanisms of HIV transcription and replication. The current classes of LRAs induce epigenetic modification, sequester transcription factors, and target the HIV Tat protein (162). Among the epigenetic LRAs, there are: histone deacetylase inhibitors, histone methyltransferase inhibitors, and bro- and extra terminal domain inhibitors. These drugs act at the HIV-1 LTR promoter and induce histone modifications at the chromatin levels, which prevent RNA polymerase from initiating transcription. There are also single-agonists LRAs like protein kinase C (PKC), that induce HIV latency reversal in model systems, which has broad impact activity and could potentially have dangerous side effects in vivo. The potential broad side effects of LRAs are one of the major challenges in employment of LRAs in the clinic for pediatric HIV treatment.

Many anti-tumor drugs approved for use in adults and children are currently under evaluation for their potential to act as LRAs in adults with HIV ( Table 3 ). Existing anti-tumor drugs are easily translatable to LRA discovery because of the shared strategy to induce epigenetic changes that promote cell cycle arrest, apoptosis, autophagy, and cell death of cancer-infected cells (163). Several LRAs have undergone testing in adult clinical trials including the histone deacetylase inhibitors, such as vorinostat, panobinostat, belinostat, and romidepsin, as well as immunomodulatory compound such as IL-15 receptor superagonist complex (N-803). While some drugs have induced HIV-1 transcription and T-cell activation, no candidates have effectively reduced the size of the virus reservoir (162). Clinical trials in adults with HIV and treated with ART in combination with romidepsin and vorinostat showed increased levels of plasma HIV RNA and cell-associated unspliced RNA in CD4+ T cells respectively. However, no significant changes in virus reservoir size were observed (164, 165). Recently, a regimen of vorinostat along with hydroxychloroquine and maraviroc was reported to increase the levels of plasma HIV RNA in a cohort of adults on ART but no change in the virus reservoir size was detected (166). In addition, upon ART interruption, no difference in time to viral rebound between the vorinostat and control groups was observed (166). Miller et al. reported that escalating doses of the investigational drug N-803, an IL-15 receptor superagonist complex (IL-15/IL-15α-Fc) was safe and well-tolerated in adult living with HIV in Phase 1 clinical trial (167). However, future investigation is still needed to determine the impact of N-803 on HIV reservoirs.

Currently, there are no clinical trials for evaluation of LRAs in pediatric HIV populations. However, several of the LRAs in clinical trials for adults have also been evaluated as anti-tumor drugs for pediatric cancer treatment ( Table 3 ). The clinical trials of vorinostat and panobinostat in pediatric cancer populations showed that the drugs were well-tolerated, suggesting the potential opportunity to safely translate these drugs into LRAs to the pediatric HIV population (166, 168). Preclinical studies in adult rhesus and humanized mouse models showed promising results in using the mimetics of the second mitochondrial-derived activator caspases (SMAC) to activate latently HIV-infected cells via the non-canonical NF-Kb pathway, in which increased levels of SIV-RNAs were observed in resting CD4+ T cells derived from tissues following SMAC mimetic (AZD5582) treatment (169). However, a more recent study by Bricker et al, reported that while the pharmacokinetics and pharmacodynamics (PKPD) study of repeated AZD5582 dosing was safe in preclinical infant rhesus macaques, the investigational drug showed weaker latency reversing activity in infant than in adult rhesus macaques, likely due to altered pharmacokinetics and less inducibility of infant CD4+ T cells (170). Therefore, additional studies in both clinical and preclinical models to investigate the impact of novel latency reversing strategies, in the context of developing immune system, on latent viral reservoir reactivation and their combination with anti-viral immunotherapeutic strategies would highly benefit the quest for pediatric HIV cure.

The eradication of persistent latently infected HIV reservoirs will likely require combinations of multiple immune-based interventions, whether alone or in combination with ART. In addition to the treatment regimen composition, dosing and timing will likely be key elements in efforts to limit initial establishment of a founder population of infected cells or to purge latently infected cells harboring replication competent HIV provirus in children with HIV.