Isotype Diversification of IgG Antibodies to HIV Gag Proteins as a Therapeutic Vaccination Strategy for HIV Infection

The development of vaccines to treat and prevent human immunodeficiency virus (HIV) infection has been hampered by an incomplete understanding of “protective” immune responses against HIV. Natural control of HIV-1 infection is associated with T-cell responses against HIV-1 Gag proteins, particularly CD8+ T-cell responses restricted by “protective” HLA-B alleles, but other immune responses also contribute to immune control. These immune responses appear to include IgG antibodies to HIV-1 Gag proteins, interferon-α-dependant natural killer (NK) cell responses and plasmacytoid dendritic cell (pDC) responses. Here, it is proposed that isotype diversification of IgG antibodies against HIV-1 Gag proteins, to include IgG2, as well as IgG3 and IgG1 antibodies, will broaden the function of the antibody response and facilitate accessory cell responses against HIV-1 by NK cells and pDCs. We suggest that this should be investigated as a vaccination strategy for HIV-1 infection.


Introduction
The development of human immunodeficiency virus (HIV) vaccines is a global health priority, particularly at a time when therapeutic vaccines are being considered as a component of a strategy for eradicating HIV infection [1]. However, the development of therapeutic HIV vaccines has been hampered by an incomplete understanding of protective immune responses that control HIV infection, vaccines. Activation of both cell types induces a diverse anti-viral response that, in particular, includes lysis of virus-infected cells by NK cells and production of type I interferons by pDCs [34,35]. Plasmacytoid dendritic cells also function as antigen-presenting cells for T-cells [36][37][38][39], including cross-presentation to CD8 + T-cells [40,41], and regulate B-cell differentiation [42].

The Role of Non-Neutralising Antibodies in the Control of HIV-1 Infection
Non-neutralising antibodies mediate their effect by activating accessory cells, which also function as antigen-presenting cells and/or elicit innate immune responses. Activation of accessory cells by IgG non-neutralising antibodies is mediated by the Fc region of the antibody binding to Fc receptors [43]. Antibody responses of this type elicited against HIV-1 proteins include antibody-dependant NK cell responses (often referred to as antibody-dependant cell-mediated cytotoxicity; ADCC) [44,45], antibody-dependant cell-mediated viral inhibition (ADCVI) [46] and phagocytic antibodies [47,48]. It is currently unclear to what extent these antibody responses are associated with control of HIV-1 infection. Thus, whilst ADCVI responses to whole virus may be associated with prevention of HIV infection after vaccination with recombinant gp120 [46], they are not associated with prevention of HIV-1 superinfection [49]. Similarly, long-term slow progression of HIV-1 infection has been associated with a wide breadth of antibody-dependant NK cell responses to regulatory/accessory proteins of HIV-1 [50], but immune escape from ADCC antibodies to envelope proteins is common [45].

Diversification of IgG Antibody Responses against HIV-1 Gag Proteins May Broaden Fc Receptor Ligation and Accessory Cell Responses against HIV-1
Antibody-induced activation of NK cells (including ADCC) results from ligation of FcRIIIa and is primarily mediated by monomeric or complexed antibodies of the IgG1 and IgG3 subclass, though complexed IgG2 and IgG4 antibodies can also bind to the 158V genotype of FcRIIIa, which confers a higher affinity of Fc binding than the 158F genotype [51,52]. Plasmacytoid dendritic cells express the activatory receptor, FcRIIa, as well as small amounts of the inhibitory receptor, FcRIIb, in about 10% of healthy individuals, but not the activatory receptors, FcRI or FcRIIIa [53][54][55][56][57]. FcRIIa plays a dominant role in phagocytic antibody responses [58] and has been demonstrated to facilitate the phagocytosis of immune complexes containing "self" or viral nucleic acids by pDCs, resulting in sensing of those nucleic acids by toll-like receptors and pDC activation [54,55,59].
Studies in patients with HIV-1 infection have demonstrated that FcRIIa is the major FcR mediating phagocytosis of IgG antibodies complexed with gp120 [47]. FcRIIa may be particularly effective in phagocytosis-induced activation of myeloid cells by immune complexes in HIV-1 infection, because, unlike other activatory FcRs (FcRI and FcRIIIa), signal transduction via the immunoreceptor tyrosine-based activation motif (ITAM) of FcRIIa does not require the FcR common -chain adaptor molecule, which is depleted by HIV-1 infection [60]. Support for this is provided by the observation that the 131H genotype of the FcRIIa gene, which confers higher affinity Fc binding to FcRIIa than the 131R genotype, is associated with slower progression of HIV-1 disease [61]. In contrast, the "high-affinity" 158V genotype of FcRIIIa has been associated with an increased risk of acquiring HIV-1 infection [62,63] and also with an increased risk of HIV-1 disease progression [62], though the methods for analysing disease progression in that study are open to criticism.

Diversification of IgG Antibodies against HIV-1 Gag Proteins to Include IgG2 Antibodies May Facilitate Ligation of FcRIIa by Complexed Antibodies
Studies of immune complex binding to FcRIIa in vitro demonstrate that all four subclasses of IgG are able to ligate the 131H genotype and, to a lesser extent, the 131R genotype of FcRIIa, especially in the form of large immune complexes [51,52]. Although the affinity of ligation of FcRIIa by IgG2 and IgG4 is less than that for IgG1 and IgG3, analyses of plasma immune complexes suggest that IgG2 antibodies play a particularly important role in the binding of immune complexes to FcRIIa. IgG2 is the most abundant IgG isotype in plasma IgG/IgM immune complexes of healthy individuals and a disease-associated increase in the ratio of IgG3 to IgG2 in the immune complexes is associated with decreased binding to Fc receptors on myeloid cells [64]. We have shown that IgG2 is much more abundant than IgG1 in FcRIIa-binding immune complexes from plasma of healthy individuals and HIV controllers, but that failure to control HIV-1 replication is associated with more abundant IgG1 in the immune complexes [48].
It is well-established that IgG2 antibodies and FcRIIa play an important role in phagocytic antibody responses against polysaccharide antigens of encapsulated bacteria [65,66]. We suggest that IgG2 antibodies also contribute to phagocytic IgG antibody responses against antigens of persistent viruses, such as HIV-1, mediated via immune complexes and FcRIIa expressed by pDCs. Targeting of viral antigens to FcRIIa on BDCA-3 + dendritic cells by IgG antibodies has been proposed as a strategy for eliciting T-cell responses against viral antigens [67]. IgG2 is the only IgG subclass capable of covalent dimerization [68], which may enhance the function of this subclass of IgG antibody in phagocytosis and/or immune complex formation. In addition, IgG2 exhibits the highest degree of resistance to proteolytic degradation [69] and may also exhibit greater resistance than IgG1 to the adverse effects of deglycosylation of the Fc region on binding to FcRIIa [70], though this was not confirmed in another study [52]. Support for our hypothesis that an IgG antibody response against HIV Gag proteins that has diversified to include IgG2 antibodies may be beneficial in the control of HIV-1 infection has been provided from studies in HIV-1 controllers or long-term non-progressors (LTNPs). Ngo-Giang-Huong et al. [71] examined plasma samples from 71 LTNPs, who had plasma HIV-1 RNA levels varying from <20 to 860,000 copies/mL and demonstrated that IgG2 antibodies to p55 and p24 were associated with lower plasma HIV-1 RNA levels. In contrast, plasma levels of IgG1 antibodies to these antigens did not correlate with HIV-1 RNA levels. We have examined plasma samples from 32 HIV-1 controllers, of whom 14 were elite controllers (plasma HIV RNA level <50 copies/mL), for IgG1 and IgG2 antibodies to HIV-1 proteins and shown that controllers had higher levels of IgG2 antibodies to Gag proteins than non-controllers and that this association was strongest in patients who did not carry the "protective" HLA-B57 allele [48]. In contrast, Banerjee et al. [72] examined serum from 16 HIV-1 controllers, of whom 13 had a plasma HIV-1 RNA level of <75 copies/mL, and demonstrated that although serum levels of total IgG and IgG1 antibodies to p24 were higher in HIV-1 controllers than patients with progressive HIV-1 disease, serum levels of IgG2 anti-p24 did not differ between HIV-1 controllers and patients with progressive HIV-1 disease. It is unclear why control of HIV-1 replication was associated with IgG2 antibodies against HIV-1 Gag proteins (p55 and/or p24) in two studies [48,71], but only with IgG1 antibodies to HIV-1 p24 in another [72]. Differences might reflect the use of Western blot assays and antigens from virus lysates in the studies by Ngo-Giang-Huong et al. [71] and French et al. [48], as opposed to ELISAs and recombinant HIV-1 proteins in the study by Banerjee et al. [72]. Furthermore, the study of HIV-1 controllers by Banerjee et al. [72] did not subgroup patients according to carriage of "protective" HLA-B alleles.
In summary, we suggest that diversification of an IgG antibody response against HIV-1 Gag proteins to include IgG2 antibodies, as well as IgG3 and IgG1 antibodies, may enhance the activation of accessory cell immune responses by NK cells and pDCs via ligation of both FcRIIIa and FcRIIa.

Isotype Diversification of IgG Antibodies to Core or Capsid Proteins of Other Persistent Viruses Is Associated with Control of Infection
Further support for our hypothesis that isotype diversification of IgG antibodies against HIV-1 Gag proteins is associated with control of HIV-1 infection is provided by evidence from patients infected by other persistent viruses. Data from patients with acute hepatitis C virus (HCV) infection suggests that IgG2 antibodies to HCV core proteins might be associated with clearance of HCV infection. Zein et al. [73] reported that all of the four patients who spontaneously cleared HCV infection had IgG2 antibodies to HCV core proteins compared with only nine of 23 patients who did not clear the infection. Furthermore, the ratio of IgG2/IgG1 HCV core-specific antibody titres was >1 in three of the four patients. In addition, studies in patients with human papillomavirus (HPV) infection demonstrated that IgG2 antibodies to capsid proteins were associated with protection from HPV disease using an ELISA and virus-like particles as antigens [74], though IgG2 antibodies could not be detected at all in another study when capsid proteins were used as antigens [75].

Regulation of IgG Antibody Isotype Diversification and the Effect of HIV Infection
Isotype diversification of IgG antibody responses occurs during the process of B-cell differentiation and maturation of the antibody response, which occurs in germinal centres of lymphoid tissue follicles following the interaction of naive B-cells with follicular dendritic cells and follicular-helper T-cells (T FH -cells) [76]. Immunoglobulin isotype switching during B-cell differentiation occurs through class switch recombination of immunoglobulin heavy chain genes, with switching to IgG2 and IgG4 occurring "downstream" of IgG3 and IgG1 [77], and results in broadening of IgG antibody function mediated by the Fc region (Table 1). Together, IgG1 and IgG2 comprise about 90% of serum IgG [78] and, therefore, exert the largest functional effect on an IgG antibody response.
Regulation of immunoglobulin isotype switching is mediated primarily by molecules expressed on, or produced by, T FH -cells [76]. The most important are the co-stimulatory molecules, CD40 ligand and inducible co-stimulator (ICOS), as exemplified by the association of immunoglobulin deficiency with deficiency of these molecules [79,80], and the cytokines, IL-4, IL-10 and IL-21, as exemplified by the restoration of immunoglobulin production by B-cells from patients with IgA deficiency or common variable immunodeficiency disorder when cultured with these cytokines [81][82][83]. The co-inhibitory molecule programmed death (PD)-1 is also highly expressed by T FH -cells, and ligation by the ligand PD-L1 has been shown to down-regulate ICOS expression and IL-21 production and possibly contribute to T FH -cell dysfunction caused by HIV infection [84]. Pro-inflammatory cytokines, such IL-2, IL-6 and IFN-, also contribute to isotype diversification of IgG antibodies, but primarily by enhancing production of IgG subclasses rather than initiating isotype switching [85][86][87][88][89] (Figure 1).  Figure 1. Isotype diversification of an IgG antibody response. IgG antibody isotype switching during B-cell differentiation in germinal centres results from class switch recombination of immunoglobulin heavy chain genes from "downstream" (IgG3 and IgG1) to "upstream" (IgG2 and IgG4) isotypes regulated by co-stimulatory molecules (CD40L and inducible co-stimulator (ICOS)) and cytokines (IL-4, IL-10 and IL-21). Pro-inflammatory cytokines (IL-2, IL-6 and IFN-) enhance immunoglobulin production with IFN- particularly increasing IgG2 production. CD4 + T-cell production of both IL-21 and IFN-is impaired by HIV infection.
While B-cell activation and increased production of total IgG is characteristic of HIV infection, driven to a large degree by pro-inflammatory cytokines [90], IgG2 deficiency is common in HIV patients [47,91], and IgG2 and IgA are less abundant in lymph node germinal centres of HIV patients than controls [92]. Indeed, serum levels of the "upstream" isotypes, IgG3 and IgG1, are increased, whereas serum levels of the "downstream" isotypes, IgG2 and IgG4, are decreased in HIV patients [90,93], suggesting an acquired disorder of B-cell differentiation and isotype diversification similar to that in patients with primary antibody deficiency disorders [94].
Data from studies of cytokine regulation of IgG subclass production by B-cells [85,86,88] and of patients with IgG2 deficiency [89] indicate that IFN-plays a particularly important role in the production of IgG2. Decreased IgG2 production in HIV patients may therefore be a consequence of both impaired B-cell isotype switching associated with T FH -cell dysfunction [84,95] and impaired IFN-production that characterises HIV-induced immunodeficiency, but is preserved in HIV controllers [6]. We provided evidence in support of this proposal from a study of antibody responses to HIV p24 in ART-treated HIV patients enrolled into a clinical trial of a recombinant DNA vaccine encoding a fowlpox virus vector, HIV Gag-Pol and IFN- [96]. Although the number of patients was small, this study provided evidence that the vaccine construct containing the gene for IFN- increased IgG antibodies to HIV p24, including IgG2 antibodies, which were associated with better control of HIV replication after ART was ceased in patients who possessed the 131H genotype of FcRIIa, which results in the highest affinity binding of IgG2 antibodies to that receptor.
It is notable that lymph node T FH -cells of patients with HIV-1 infection exhibit greater reactivity with Gag proteins than Env proteins [97]. Dysfunction of T FH -cells associated with HIV-1 infection [84,95,97] may therefore contribute to limited isotype diversification of IgG antibodies against HIV-1 Gag proteins.

Potential Strategies for Enhancing Isotype Diversification of IgG Antibodies to HIV-1 Gag Proteins
Therapeutic modulation of the isotype of vaccine-induced IgG antibodies is not an established procedure in humans, but has been achieved in dogs with a saponin-adjuvanted Leishmania vaccine [98]. Preliminary data from patients with HIV-1 infection suggest that IFN- might enhance vaccine-induced IgG2 antibodies to HIV-1 Gag proteins [96], and this potential approach to therapeutic vaccination should be considered further. Finally, inhibition of immune activation in HIV-1 patients by PD-1 blockade might also have beneficial effects on T FH -cell function [84] and antibody responses [99], and examination of IgG antibody isotype diversification might be examined in clinical trials of therapies that block the PD-1/PD-L1 pathway.

Conclusions
We propose that enhancing isotype diversification of IgG antibody responses against HIV-1 Gag proteins during vaccination, to include IgG2, as well as IgG3 and IgG1 antibodies, may result in an IgG antibody response that facilitates the accessory cell responses of NK cells and pDCs to elicit both ADCC responses by NK cells, as well as phagocytosis of complexed antibody by pDCs and a pDC-dependant antiviral response (Figure 2). Further experimental evidence is required to strengthen our hypothesis. In particular, studies are needed to establish that IgG2 antibodies inhibit HIV-1 replication and are not just a marker of Th1 responses. However, at a time when new approaches to the development of HIV vaccines are needed [2], we suggest that consideration should be given to vaccination strategies that will enhance isotype diversification of IgG antibodies against HIV-1 Gag proteins.

Figure 2.
A diagrammatic representation of how isotype diversification of IgG antibodies against HIV-1 Gag proteins might enhance anti-viral accessory cell responses against HIV-1 infection. It is proposed that IgG antibodies bind to HIV-1 Gag proteins expressed on the surface of cells infected by HIV-1, including resting CD4 + T-cells [9]. Activation of natural killer (NK) cells is elicited by "downstream" IgG isotypes (IgG3 and IgG1) via FcRIIIa. "Upstream" IgG isotypes (IgG2 and possibly IgG4) may also contribute to NK cell activation by ligating FcRIIIa, particularly in individuals carrying the 158V genotype. However, it is proposed that multimeric IgG2 antibodies primarily broaden the function of the antibody response by enhancing phagocytic activity against Gag proteins associated with HIV-1 RNA, as a consequence of the functional characteristics of IgG2 (see Table 1), which activates plasmacytoid dendritic cells (pDCs) via FcRIIa. Activation of pDCs leads to the production of IFN-which facilitates NK cell responses and induces the production of interferon-stimulated genes (ISGs) and to antigen presentation and/or stimulation of B-and T-cells (see text). HIV-1 infection impairs diversification of an IgG antibody response to "downstream" isotypes.