**3. Immune response**

HIV-1 infection has a unique pathogenesis and degree of biological complexity unparalleled by other viruses. This biological complexity involves a wide range of immune cells and mechanisms, with the initial CD4 and CD8 T cell response, viral entry, and establishment of the latent reservoir reviewed in this section. Additionally, autologous neutralizing antibodies, broadly neutralizing antibodies (BnAbs), and extensive B cell dysfunction are important aspects to the immune response, but details are given in Sections 3.1–3.3.

In the initial stage after HIV-1 exposure but before infection, there are competing "HIV viral quasi-species" found in the donor secretions, vaginal mucosa of the recipient, and systemic component [11]. In the majority of infections (>75%), one variant referred to as the transmitted founder invades past the mucosal lumen to the stroma and travels to the local lymph nodes. (In 20% of infections, there are multiple variants involved in establishing systemic infection.) This transmitted founder has little genetic diversification. Here, at the lymph node, the CD4 T cells disperse the infection and exponential viral amplification, and systemic spread occurs [11].

Once systemic spread from the CD4 T cells is established, the CD8 T cells partially suppress the peak viral load after 30 days of infection. HIV rapidly evades the immune system and mutates to alter the epitope-human leukocyte antigen (HLA) binding. Over the next few weeks to months, the one viral variant becomes more like quasi-species version after exposure and diversifies its viral genome, due to the errorprone reverse transcriptase machinery adding different genetic codes to each infected cell [11, 12]. These viral variants may differ up to 40% of amino acids [13].

Though HIV-1 infection progresses in this pattern discussed in the last two paragraphs, understanding the specific mechanism of the virus entry is important aspect of the immune response. This mechanism begins with HIV envelope (Env) protein, gp120, binding CD4, and a cell surface protein receptor present on multiple types of immune cells. This gp120/CD4 interaction induces a conformational change, allowing gp120 to then bind to a coreceptor, CCR5, or CXCR4 [14–16]. Once coreceptor has been activated, gp41, another HIV-1 envelope protein, changes configuration to form a trimer-of-hairpins and fuses HIV-1 to the cell membrane, finalizing the entry [14].

The coreceptor of CCR5 or CXCR4 is of particular importance because this determines their susceptibility to infection. HIV-1 virus has two main "tropic" species, macrophage-tropic or M-tropic, and T-cell-tropic or T-tropic viruses [15, 16]. M-tropic strains are noted to be more preferentially transferred and utilize the CCR5 coreceptor, while T-tropic strains target the CXCR4 coreceptor [16]. Interestingly, there are some unique intermediate viruses referred to as "dual-tropic," meaning they can infect cells with either CXCR4 or CCR5 [15, 16]. Some drugs in ART regimens and in clinical trials involve mechanisms to antagonize the CCR5 or CXCR4 coreceptors

and inhibit viral fusion inhibitors [14, 16]. Though drugs are effective, there are several associated problems, including multidrug resistance, adverse effects, and high cost [14]. Further research into understanding the mechanisms of HIV-1 entry and potential steps to terminate the infection may prove to be advantageous.

One of the major challenges of this virus is the early formation and somewhat permanent establishment of latent reservoir of infected CD4+ T cells. HIV-1 preferentially targets CD4+ T cells and then quickly reduces the memory CD4+ T cells in gut-associated lymphoid tissue in the first 4–10 days [17]. There are some studies that suggest that the initial prime target of the virus is epithelial Langerhans cells and dendritic cells during HIV-1 vaginal transmission, instead of the CD4 lymphocyte [11].

Regardless of the HIV-1 initial target, the impact of memory CD4+ T cell infection is significant because this establishes the latent reservoir and allows the virus to persist even with ART [18]. Utilizing nonhuman primate (NHP) simian immunodeficiency virus (SIV) studies, the latent reservoir is established within the first three days of infection, even before the virus is detectable [11].

Additionally, besides the establishment of the latent reservoir, there is another deleterious effect affecting CD4+ T lymphocytes in gastrointestinal mucosa. The Th17 subset of CD4+ T cells has an established role for maintaining mucosal integrity in the gastrointestinal tract. When these cells are infected, they no longer maintain a tight barrier or prevent gut bacterial products from invading the bowel wall [19]. This leads to microbial translocations, chronic immune activation, and shifts in microbiome [19–22].

#### **3.1 Autologous neutralizing antibodies**

Though the B cell response is less than optimal, as discussed in more detail in Section 6, the immune system is eventually able to produce autologous strainspecific neutralizing antibodies to the transmitted founder virus and the viral escape mutants. This occurs approximately three months to a year after infection [11]. These neutralizing antibodies are estimated to neutralize approximately 50% of the diverse strains of HIV-1 and only occur in half of patients with HIV-1 infections [12]. While these antibodies are ultimately overwhelmed by the continued viral evolution over the subsequent years, it is an important stepping stone for some individuals to progress generating broadly neutralizing antibodies [11], as reviewed in the next section.

#### **3.2 Broadly neutralizing antibodies**

Broadly neutralizing antibodies (BnAbs) are defined as antibodies with the ability to neutralize diverse isolates of HIV-1 and represent a potential avenue for prophylaxis and therapy [23]. This class of antibodies have high levels of mutations in rare, low-affinity naive B cell receptors [24], and these B-cell receptor (BCR) display atypical structural and binding characteristics that the immune system usually negatively selects against during B cell development [24, 25]. Some of the features of BnAbs are similar to those of autoreactive antibodies, indicating that they may evade immune tolerance mechanisms [26, 27]. Through the chronic viral replication, BnAbs are generated due to the extensive affinity maturation in germinal centers [27, 28].

BnAbs are an interesting and important phenomenon of the HIV-1 immune response, because only 10–20% of people with HIV-1 infections produce these antibodies after many years of infection [24, 25]. These individuals usually take approximately two to three years to produce BnAbs and are sometimes referred to as "elite neutralizers" [29]. While the BnAbs are able to neutralize many strains of HIV-1 variants, they ultimately are futile at controlling the host's infection because of years of sustained viremia [24].

Progress in the identification of BnAbs as well as the study of structural properties has significantly advanced the field, due to its application as potential therapy. To identify BnAbs, researchers can isolate HIV-1 Env-reactive memory B cells from multiple sources including antigen-specific B cell sorts, from plasma cell sorts, and from clonal memory B cell cultures [25].

The structural studies of BnAbs contribute to our understanding of the immune response to HIV-1 infection and its progression. Generally speaking, BnAbs perforate the glycan shield of the HIV Env trimer in five regions, likely dismantling Env function [23]. This massive glycan shield of the HIV Env trimer concealing the antigenic target is a major obstacle for the humoral immune system to produce antibodies that can neutralize HIV-1 variants [30].

Of these regions in the HIV Env trimer, the V2 apex is one of the most important because of its role in maintaining the metastability of the Env spike, which influences the CD4/CCR5 conformational changes. The CD4 binding site (CD4bs) is the primary receptor for HIV and exposes CCR5 after activation. Interfering with the V2 apex may prevent viral penetration. BnAbs targeting the V2 apex are considered a potent antibody but limited in breadth (less than 70%) and display incomplete neutralization (<100%) [23]. Further research is needed to elucidate this field.

## **3.3 B cell dysfunction**

During acute HIV infection, multiple immune components lead to extreme B cell dysfunction. This is namely due to of polyclonal activation, hypergammaglobulinemia, nonspecific plasmablast surge, impaired memory and naïve B cells, and B cell exhaustion [28, 31].

Through polyclonal activation, B cells terminally differentiate into plasmablasts (cells rapidly produced in early antibody response to generate antibodies [32]) and plasma cells approximately a week after infection [11]. Polyclonal activation has been studied and shown to be elicited *via* multiple pathways directly from serum cytokines and indirectly from HIV Nef protein [28]. The polyclonal activation leads to a state of hypergammaglobulinemia [31].

Interestingly, though there is a state of hypergammaglobulinemia, the plasmablasts increase to comprise only up to 13% of circulating B cells. This response is not pathogen-specific since only 1.5% or less are HIV-specific and unable to neutralize the virus. In other viral infections like RSV and dengue virus, plasmablasts increase to comprise 30% of total lymphocytes, with the majority being pathogen specific [11, 33].

During HIV-1 infection, there is an increase in circulating antibodies and increase in activity of B cells, but a disruption in the microgenerative environment impacting memory and naïve B cell subsets [28]. This decline in circulating memory B cells is of particular importance and could be classified as a marker of disease progression, as they are linked to CD4+ T cell population numbers [34]. The low levels of memory B cells lead to the characteristic opportunistic infections, namely *Pneumocystis carinii* and *Cryptococcus neoformans* [34].

Among the B cell dysfunction category is B cell exhaustion, only recently described in 2008 in HIV. B cell exhaustion refers to the decreased ability to

proliferate in response to *de novo* stimuli. HIV infections cause this exhaustion in a specific subset of B cells that are tissue-like memory B cells. This subset of cells has increased expression of multiple inhibitory receptors (CD22, CD72, and LAIR-1) as compared to normal resting memory and terminally differentiated B cells. Other unique inhibitory receptors to B cell exhaustion are also being investigated. Overall, B cell exhaustion is similar to CD4 and CD8 T cell exhaustion, but B cell exhaustion has proven to be more challenging to study since the experimental science is less direct as compared to T cell assays [31].
