**4. Variety of immune responses: progressors, immunological nonresponders, long-term nonprogressors, and elite controllers**

One challenging aspect of HIV-1 infections is that there are a wide range of potential immune responses. There are four categories used to describe an individual's immune response to HIV-1: HIV-1 natural progressors, immunological nonresponders (INR), elite controllers, and long-term nonprogressors. Each category is described in detail below.

HIV-1 natural progressors refer to a typical response to an HIV-1 infection. The timeline for this response is not clearly defined but most likely reflects an intermediate progression where AIDS develops 3–10 years after seroconversion [35]. If these patients were to receive ART, the progression to AIDS would be less likely or potentially very slow and gradual.

Immunological nonresponders (INRs) have not been universally defined, but the most general accepted classification of INR is a patient who does not meet a specific CD4+ T cell count level or a specific percentage CD4+ T cell increase over baseline after a certain length of ART. The literature values for these specific levels and percentages vary widely with the CD4+ T cell count range of 200–500 and percentages from over 5–30%. The length of ART also changes from 6 to 144 months. Though the CD4+ T cell does not increase as it should, the viral load is suppressed. Given the inconsistent definition across studies, this subset is approximated at 10–40% of people with HIV-1 infections. This subset of people with HIV-1 infections is more likely to have morbidity and mortality from AIDs and non-AIDs conditions, because their immune systems are significantly dysfunctional [22].

Elite controllers and long-term nonprogressors represent those with immune responses that suppress HIV-1 viral load naturally without ART. These categories are differentiated by the degree of viral load suppression [36]. Elite controllers suppress the HIV-RNA values to less than 50 copies per milliliter, while long-term nonprogressors suppress the HIV-RNA to less than 5000 copies per milliliter [35].

Elite controllers are rare, estimated to be 0.1–1% of all people with HIV-1 infections but represent a functional cure. Understanding this population may lead to greater understanding of successful immune responses against HIV-1 [18]. As described in Loucif paper, elite controllers maintain suppressed viral loads most likely due to a combination of the following factors: high-quality and polyfunctional CD8+ T cells, memory B cell responses, preserved memory and pTfh CD4+ T cells, lack of natural killer (NK) activation, preserved plasmacytoid dendritic cell counts, and preservation of gut mucosal immunity [8].

The polyfunctionality of CD8+ T cells is a key differentiating factor between elite controllers and progressors. It has been found that the CD8+ T cells in elite controllers are able to degranulate properly and release perforin, granzyme, and cytokines

(interferon-gamma, tumor necrosis factor-alpha, interleukin-2, and macrophage inflammatory protein-1beta) [36]. It is believed that the functional CD8+ T cell response is directly linked to disease progression [36].

Long-term progressors display similar characteristics as elite controllers, primarily the polyfunctional T cells [17]. They maintain high levels of CD4+ and CD8+ T cells without ART therapy. Approximately 5% of the total HIV population are long-term nonprogressors [21, 35].

#### **4.1 Disadvantages for elite controllers and long-term nonprogressors**

While the immune responses of elite controllers and long-term nonprogressors control the viral load in general, there are some downsides that these groups face. These patients can decline at any time, despite having long periods of naturally suppressed viral loads. While it is challenging to estimate the number of regressions in a small population of those with HIV-1 infections, it is believed that about 25% of elite controllers decline [37].

Researchers compared elite controllers who lost the ability to suppress the virus against the "persistent" elite controllers. The "persistent" elite controllers had low viral diversity, low HIV-DNA concentrations, overall lower inflammation, decreased immune activation, and proinflammatory cytokine concentrations. It was also found that the high Gag-specific T-cell polyfunctionality was no longer present in the individuals who lost viral control [37].

Though elite controllers represent a functional cure to HIV-1 infections, it is worth noting that this subset of patients is susceptible for hospitalizations of all causes (as compared to patients with HIV-1 infections on ART). The majority of these hospitalizations were due to cardiovascular and psychiatric diseases [36]. It is believed that a subset of elite controllers has this increased risk of hospitalizations and adverse effects because there is a persistent immune dysfunction driving the pathology [19].

## **5. Pediatric immune response**

In addition to the various categories of immune responses, there is also a subset of young patients who acquire the infection perinatally or from breastfeeding from an HIV-1 positive mother. This occurs in part due to the large number of reproductive age women with limited access to ART and birth control in low-income countries. These patients have a different timeline than the standard adult, in terms of both immune response and overall disease course, and represent a significant public health crisis in low-income countries.

The timeline of the immune response in a pediatric patient begins with an infant with HIV-1 with high titers of passively transferred maternal neutralizing antibodies until three months of age. After this point, the neutralizing antibodies decrease but increase at 12 months, meaning that the infant is able to produce this antibody type. Some of these young patients then produce broadly neutralizing antibodies much earlier in infection, with diverse epitope specificities, and higher breadth and potency than that of adult patients [29]. One broadly neutralizing antibody (BF520.1) studied was noted to have limited somatic hypermutation and an absence of insertions and deletions, unlike the studies performed on adult antibodies. Given these core differences, the pediatric BnAbs are thought to be derived from different pathways than those produced in adults [29].

The overall disease course in a pediatric patient is unique because of a faster progression to AIDS [29] and a higher risk for neurocognitive deficits [38]. Without ART, children progress to AIDS within a year, as compared to adults taking a decade to progress [29]. These patients are more likely to have neurodevelopmental and neurocognitive disorders as compared to patients who acquired HIV-1 infection as adults. Given the rapid growth of nervous system in early infancy and childhood, understanding how HIV-1 infections impact childhood development is important. Pediatric patients are more likely to have physical brain damage from the inflammation and multinucleated giant cells in the cerebral cortex. The main manifestation of this impaired neurocognition is limitations in language function [38]. The studies of neurodevelopmental and neurocognitive effects on HIV-1 infection in pediatric patients are limited but represent a growing field of interest given the number of young patients in low-income countries [38].

Notably, 53% of untreated children with HIV-1 die by two years of age in sub-Saharan Africa. Before three years of age, this statistic changes to 75% children with HIV-1 dying [9]. Because of two modes of vertical transmission with HIV-1 infection occurring perinatally (in utero or intrapartum) and through breastfeeding, these groups of children have been assessed separately and identified that the children infected perinatally are at higher risk of death (60%) as compared to children infected through breast milk (36%) [9].

Given pediatric patients' immature immune systems and progression to AIDS, more research regarding acquisition prevention in these patients as well as funding is needed to combat this public health crisis in low-income countries.

## **6. Select clinical trials**

Despite several decades of research, vaccine development, and clinical trials, currently, there is not any effective vaccine to prevent acquisition of HIV-1 infection. When HIV-1 was initially identified as the causative agent for AIDS in 1983–1984, researchers believed that a prophylactic vaccine would be generated within two years. This two-year estimate drastically underestimated the challenges and biological complexity of HIV-1 and illustrated the fact that HIV-1 is unlike any other viral disease that has a vaccine [4].

Though most clinical trials have found no efficacy, one clinical trial referred to as RV144 had unexpected success. This trial was controversial before it even began because it was believed to have a high likelihood of failure, given the early-phase clinical trials assessing the immunogens used in this trial. The initial data found the vaccine components were poorly immunogenic in isolation. Regardless of whether they were administered alone or in combination, there was only modest T-cell and humoral responses with no virus neutralization. However, the phase 3 trial proceeded in part to study a heterologous prime-boost strategy [39].

RV144 was the first trial that showed that any vaccine could induce protection against HIV-1 infection. Despite all odds, this vaccine had 60.5% efficacy in the first year [11] and decreased to 31.2% efficacy at 42-month post-vaccination [4]. Though the efficacy decreased significantly by three years, simulated studies believe that even if the vaccine had 50% efficacy for two years, it would have a significant impact in high prevalence areas [24].

As the only trial in humans with any efficacy, researchers had great interest in investigating what immune correlates were associated with protection against infection. The immune correlates for HIV-1 infection are unique inherently, given that the virus is never cleared naturally, but there is some type of protection against infection not yet understood. The immune correlates were identified as formation of non-neutralizing IgG against the V1/V2 region of HIV-1 Env, antibody-dependent cellular cytotoxicity in patients with low IgA, and Env-specific polyfunctional CD4+ T cells [4, 11, 24]. The mechanistic rationale behind how HIV-specific nonneutralizing antibodies protected against HIV-1 acquisition is not well understood and controversial [40].

In an effort to replicate RV144's success, a similar trial referred to as Uhambo or HVTN 702 was designed. There were clear differences between the two trials: RV144 had been conducted in Thailand in 2009, testing a recombinant Canarypox vector prime followed by two injections of a recombinant gp120 boost. HVTN 702 was conducted in South Africa with the same vector prime, similar protein boost but slightly different adjuvant, and different envelope sequences [24]. Investigators chose to change the envelope sequences to reflect the locally circulating HIV-1 variants in South Africa [41].

Ultimately, HVTN 702 was unsuccessful and terminated due to lack of efficacy in 2020 [41]. There was no significant production of the V2 loop antibodies deemed to be the critical immune correlate in RV144. This trial did result in high levels of binding antibodies, antibody-dependent cellular phagocytosis, and antibodydependent cellular cytotoxicity activity, but overall no efficacy [42]. Perhaps, this lack of efficacy is due to the vast genetic diversity of the Sub-Saharan African with clade C, the difference in host genetic factors, or other indeterminate factors due to clinical trial differences as discussed by Gray et al. [41].

As previously discussed, the immune response to HIV-1 infection is intricate and complex with multiple stages of infection and potential responses.

### **7. Broadly neutralizing antibodies in passive immunization trials**

Given the intricate method, the immune system forms BnAbs; immunization to induce BnAbs is proving to be exceedingly difficult. As previously reviewed in Section 5, the natural production is several years into infection and not clear why only a subset of people with HIV-1 infections generate these antibodies. The mechanism behind their evolution is also yet to be fully understood [25].

Before reviewing the vaccine strategies for BnAb induction, it is important to note that there have been clinical trials with passive immunization using VRC01, an IgG1 BnAb against the Env CD4 binding site. *In vitro* studies revealed that this BnAb has wide coverage against HIV-1 subtypes B and C. These trials were HVTN 704/HPTN 085 in the US, Peru, Brazil, and Switzerland and HVTN 703/HPTN 081 in South Africa, Zimbabwe, Malawi, Botswana, Kenya, Mozambique, and Tanzania, and ran from 2016 to 2018. The ultimate goal of these trials was to investigate if VRC01 is capable to preventing HIV-1 acquisition [43].

The results published in 2021 indicated that this BnAb was unable to prevent overall HIV-1 acquisition, but that in VRC01-sensitive HIV-1 isolates, BnAb prophylaxis was effective [43]. The VRC01-sensitive HIV-1 isolates were only ~30% of the strains in circulation [44]. These results suggest that the VRC01 suppressed early circulating strains, but the immune system eventually lost to evolving resistant viral variants. It is likely that a combination of BnAbs is necessary to prevent viral escape [43].

Additionally, "bispecific" and "trispecific" BnAbs were developed and tested in phase 1 clinical trials [43]. Bispecific or trispecific BnAbs have two or three different specificities in a single molecule. This unique class of BnAbs may lead to increased neutralization breadth and limit viral escape [45].

While studying how efficacious are BnAbs, it is important to assess their behavior *in vitro*, the administration of BnAbs *via* intravenous therapy is not a feasible drug delivery system for large populations. It may also have limited use if administered in combination of BnAbs in high-risk groups, but overall this is not feasible or sustainable method for HIV-1 prevention [44].
