To determine the effect of vaccination on HIV replication after ceasing ART, plasma HIV RNA levels were measured weekly over 20 weeks. The relationship between plasma levels of IgG2 anti-p24 at week 0 and HIV replication after ceasing ART was examined by dividing antibody levels for each group into tertiles and comparing the time-weighted increase in plasma HIV RNA level over 20 weeks for each tertile. As shown in Table 1, the time-weighted increase in plasma HIV RNA levels was lowest in patients who belonged to the highest tertile of IgG2 anti-p24. However, a similar association was also observed for IgG1 antibodies to gp41, which was not encoded by the rFPV vaccine. As IgG2 anti-p24 correlated with IgG1 anti-gp41 at week 0, adjusted analyses were attempted but could not further delineate independent effects of IgG2 anti-p24 and IgG1 anti-gp41 tertiles at baseline on time-weighted increase in plasma HIV RNA levels.
To further investigate the role that IgG2 antibodies might have had in controlling HIV replication after the ART was ceased, analyses were undertaken to examine the relationship of IgG2 anti-p24 and FcγRIIa genotype on HIV replication. As shown in Fig. 3, plasma HIV RNA levels over 20 weeks were lowest in patients from the full construct group who had both an IgG2 anti-p24 band intensity of more than 200/mm2 and the FcγRIIa HH or RH genotype (n = 3) compared with patients who had one or neither of these characteristics (n = 6). At the 20-week time point, plasma HIV RNA levels were lower in patients with both an IgG2 anti-p24 band intensity of more than 200/mm2 and the FcγRIIa HH or RH genotype compared with other full construct group patients (P = 0.002) and other patients from all groups (P = 0.0001).
We also examined the effect of ceasing ART on the plasma levels of IgG2 and IgG1 antibodies to a vaccine-encoded antigen (p24). As shown in Fig. 4, the full construct and partial construct/placebo groups did not differ when plasma levels of IgG2 anti-p24 and IgG1 anti-p24 were compared at week 9 (P = 0.75 and 0.93, respectively) or week 20 (P = 0.56 and 0.94, respectively).
We have tested the hypothesis that control of HIV replication after cessation of ART in HIV-infected patients who received a course of vaccinations with a rFPV vaccine that encoded both HIV Gag-Pol and IFN-γ was associated with IgG2 antibodies to a Gag-encoded protein (p24) and possession of the ‘high-affinity’ FcγRIIa HH or RH genotypes. Although the number of patients assessed was small, we were able to show that those patients who received the full construct vaccine had higher plasma levels of IgG2 antibodies to p24 prior to the cessation of ART (week 0) and that at this time the highest IgG2 anti-p24 responses were associated with the lowest time-weighted increase in plasma HIV RNA level after cessation of ART. In addition, we demonstrated that patients who had both vaccine-induced IgG2 anti-p24 and ‘high-affinity’ FcγRIIa genotypes had the lowest plasma HIV RNA levels after ART was ceased.
Plasma levels of IgG1 antibody to p24 and to gp41 (an antigen not encoded by the rFPV vaccine) at week 0 were also higher in patients who had received the full construct vaccine. Indeed, at week 0, plasma levels of IgG2 anti-p24 correlated with levels of IgG1 antibodies to both p24 and gp41. In contrast, IgG2 anti-gp41 levels were not increased in the full construct vaccine group at week 0 (Fig. 1b). These findings suggest that the IFN-γ produced at the site of vaccination enhanced IgG1 antibody responses to both vaccine-encoded and nonencoded antigens but only induced isotype switching to IgG2 antibodies for an antigen encoded by the rFPV vaccine. Like IgG2 anti-p24, higher serum levels of IgG1 anti-gp41 at week 0 were associated with a lower time-weighted increase in plasma HIV RNA after cessation of ART (Table 1). We were unable to determine the relative effects of IgG2 anti-p24 and IgG1 anti-gp41 on HIV replication after ceasing ART because of small patient numbers. It is therefore not possible to exclude an effect of IgG1 anti-gp41 alone or in combination with IgG2 anti-p24. However, it should be noted that studies in patients with primary HIV infection suggest that IgG anti-gp41 has little impact on HIV replication .
Plasma levels of IgG2 and IgG1 anti-p24 progressively increased after cessation of ART, presumably reflecting the increase in HIV replication in most patients . However, there was no difference in plasma levels of these antibodies between the full construct and partial construct/placebo groups at weeks 9 or 20. This suggests that when HIV replication is suppressed, IFN-γ at the site of vaccination facilitates production of IgG2 antibodies that may be associated with suppression of HIV replication when ART is ceased but that in the presence of generalized immune activation after resumption of HIV replication, IgG2 antibodies induced by polyclonal B-cell activation have no additional effect.
Human IgG2 antibodies are associated with type 1 ‘helper’ T-cell (Th1) responses in experimental cryptococcal infection  and systemic lupus erythematosus (SLE) . This may also be the case in HIV infection because Martinez et al. demonstrated that the combination of a strong Th1 CD4+ T-cell response to p24 and a strong IgG2 antibody response to gp41 predicted long-term nonprogression of HIV infection better than any other marker, including the genetic markers examined in that study. The nature of the association between IgG2 antibodies and Th1 responses has not been established but it might reflect functional characteristics of the Fc region of IgG2. The Fc region of IgG2 activates the complement system poorly  and mediates its effect predominantly by activating FcγRIIa (CD32a) , which is a low-affinity FcγR that is only activated by multimeric antibodies, particularly in immune complexes [29,30]. Of note, IgG2 is the predominant IgG subclass in circulating immune complexes of healthy individuals .
FcγRIIa is only present in higher primates and is expressed on the surface of cells that function as antigen-presenting cells (APCs) and/or phagocytes such as conventional dendritic cells, monocytes, macrophages, platelets, endothelial cells, neutrophils and eosinophils . It is also expressed on plasmacytoid dendritic cell (pDC), the major cell type involved in antiviral immune responses, in which phagocytosis of antigens in immune complexes is one means by which antigens are acquired and presented on class II major histocompatibility complex (MHC) molecules to CD4+ T cells [32–34]. Studies in patients with SLE have shown that pDC activation and production of interferon-alpha (IFN-α) is induced by complexes of DNA and IgG anti-DNA binding to FcγRIIa leading to transportation of the immune complexes to endosomes in which CpG DNA binds to TLR9 . This results in the production of proinflammatory and Th1 cytokines as well as IFN-α. A similar mechanism of phagocytosis via FcγRIIa and intracellular transportation to endosomal TLR7 has been described for immune complexes of Coxsackievirus RNA and antibody . It is therefore possible that HIV antigens are processed by pDC in a similar way. FcγRIIa may be particularly effective in this process because, unlike FcγRI and FcγRIIIa, it is not functionally impaired by HIV infection .
Our argument that IgG2 antibodies against p24, an internal protein of HIV, might be enhancing opsonization of HIV may appear to be counter-intuitive. However, it has been clearly demonstrated that antibodies to p24 are associated with slower progression of HIV disease [38,39], though the mechanism is unclear. IgG2 antibodies are a major component of the antibody response to carbohydrate antigens and facilitate opsonization of encapsulated bacteria by binding to FcγRIIa [40–42]. An IgG2 antibody response to glycoproteins of the HIV envelope might therefore be an explanation for the association between nonprogressive HIV disease and IgG2 antibodies to HIV gp41 [13,14]. In this study, we evaluated IgG2 antibodies to p24 because the rFPV vaccine, which was designed to enhance T-cell responses, encoded only HIV Gag-Pol antigens. There are also other characteristics of IgG2 that might enhance a protective immune response against antigens of HIV. These include the ability of IgG2 to form covalent dimers, thereby enhancing FcγR binding , and resistance to the decreased binding of FcγRIIa to IgG antibodies caused by deglycosylation of the Fc region of IgG molecules , which is an effect of HIV infection .
In summary, we have demonstrated in a small randomized controlled trial that vaccination with rFPV that encoded HIV Gag-Pol and IFN-γ resulted in the production of IgG2 antibodies to a vaccine-encoded protein (p24) in five of nine (54.5%) patients and that such antibodies were associated with control of HIV replication in individuals who possessed the ‘high-affinity’ FcγRIIa HH or RH genotypes. We suggest that further attention should be paid to the role of B-cell immunoglobulin isotype switching, IgG2 antibodies and FcγRIIa in the control of HIV infection. Furthermore, inclusion of the IFN-γ gene in DNA vaccine constructs might be a means of enhancing switching to IgG2 antibody production.
The authors wish to thank Ibrahim Fleyfel for technical assistance and Professor Ian James for advice on statistical analysis of the data.
Author contributions: M.A.F. devised and undertook overall supervision of the study and wrote the first draft of the manuscript. S.T. and A.L. undertook the antibody assays. S.E., A.D.K. and L.D.W. established the original clinical trial and provided patient samples and funding. M.G.L. and S.F. undertook analysis and presentation of the data. All authors reviewed and approved the final version of the manuscript.
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