Introduction.

Peptide-loaded MHC class I molecules are expressed on the surface of all nucleated cells, while MHC class II molecules are expressed on the surface of professional antigen presenting cells. For the clearance of intercellular pathogens during the course of an infection, foreign antigens specific to the pathogen are processed and presented on MHC class I and/or MHC class II molecules [1]–[3]. The presentation of foreign peptides by MHC class I and MHC class II molecules on the surface of cells induce epitope specific CD8+ and CD4+ T-cells. These antigen-specific T cells are then recalled during re-exposure to the pathogen.

Antigen processing and presentation is a complex process involving many proteins working in a defined order. Although there are differences in the proteins required for MHC class I and MHC class II processing and presentation, antigens processed through one pathway can also be presented by the other pathway [4].

MHC class I processing involves many proteins such as ubiquitination proteins, chaperone proteins, loading and transporter proteins, and proteases including the proteasome complex. Endogenous antigens within the cytoplasm are mainly processed by proteasomes [5]–[12] before transportation into the endoplasmic reticulum via the transporter associated with antigen processing. Further trimming of these peptides occurs within the ER before the peptides can be loaded onto the MHC class I molecules [13]. The 8–10 amino acid epitope bound to an MHC class I molecule is then transported to the cell surface.

MHC class II processing of exogenous and endogenous antigens occurs in the endosomal/lysosomal compartment. The antigens can enter the endosomal compartment through endocytosis, phagocytosis, or by autophagy [14]. The antigens are processed by cathepsins and other proteases present in endosomes/lysosomes. There are several cathepsins some of which are cell-type specific. Cathepsins L and S are cysteine proteases while cathepsin D is an aspartic protease. These enzymes cleave endocytosed antigens and generate peptides for MHC class II binding as well as remove the invariant chain chaperone [15], [16]. The processed antigen is presented on the cell surface as a 12–15 amino acid epitope bound to an MHC class II molecule [2], [17]–[20].

Intracellular pathogens have evolved multiple mechanisms to avoid the host's immune response and one principal mechanism is to disrupt or prevent antigen processing and presentation. This can potentially negate or alter the epitope repertoire of foreign epitopes bound to MHC class I or MHC class II molecules on the cell surface [8], [21]–[26]. Apart from directly interacting with the antigen processing and presentation machinery, the biochemical properties of antigens such as disulfide bonds and glycosylation may also influence antigen processing [27], [28]. Disulfide bonds and folding may impact the ability of specific proteases such as the proteasome to proteolytically cleave folded antigens [28]. The processing of exogenous and endogenous glycosylated protein antigens can be impacted by the presence of terminal mannose and fructose residues, which could predetermine the initial processing of glycoprotein antigens within lysosomes and endosomes [29], [30].

The interaction of either mannose or fructose with the appropriate lectins on the cell surface induces the formation of phagosomes and engulfment of the pathogen or antigen for delivery into the phagolysosome for proteolytic degradation and presentation on MHC class I and class II molecules [31]. HIV-1 envelope (Env) is heavily glycosylated with mannose [32], [33], and studies using immature dendritic cells have demonstrated that the envelope protein is capable of being processed in the phagolysosome [34]. Endogenous glycoproteins generated within the cell can be cannibalized by lysosomes through a mechanism known as autophagy [14] and the proteolytically cleaved peptides can be presented by both MHC class I and class II molecules.

Elucidating how antigens are processed and presented is critical for the design of vaccines. The recent RV144 Phase III clinical trial in Thailand using a canary pox vector ALVAC (vCP1521), carrying HIV clade B and circulating recombinant form (CRF)01_AE gag, pro and env genes, and AIDSVAX®B/E (genetically engineered gp120) protein as the vaccine demonstrated a modest efficacy of 31.2% [35]. An analysis for correlates of risk suggested that, among cellular assays, the production of cytokines after stimulation of PBMC from volunteers was inversely correlated with infection rate, although statistically the effect was less robust than the correlates identified for IgG binding to a conformational V1/V2 epitope(s) and Env-specific IgA [36]. Preliminary immunological analysis of RV144 vaccinee samples demonstrated that the vaccine induced a very poor direct ex vivo CD8+ T cell response and that both the CD4 cellular and the humoral immune responses were biased towards the HIV-1 gp120 Env antigen. In order to determine if the reason for the low Env-specific CD8+ T cell response compared to the CD4+ T-cell response was because of the efficiency of antigen processing, this study examined the susceptibility of Env-A244 gp120 protein, one of the components of the bivalent gp120 protein subunit boosts used in the RV144 trial. Env-A244 gp120 protein was subjected to cleavage by purified proteasomes or cathepsins or cathepsins followed by proteasomes and the peptides generated were characterized by mass spectrometry. The peptides generated were examined for their functional activity. We have identified a peptide fragment derived from cathepsins D and K degradations of Env-A244 gp120 (Env-A244) protein containing the sequence DKKQKVHALF in the V2 loop of gp120 that induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines derived from RV144 vaccinees suggesting a possible link between proteolytic processing and induction of vaccine-specific CD4+ T helper cells.

Abstract.
Background.

Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response.

Methods.

In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers.

Results.

Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial.

Conclusions.

Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees.