Cross-Clade CD8 T-Cell Responses to HIVIIIB and Chinese B' and C/B' Viruses in North American and Chinese HIV-Seropositive Donors

HIV variation presents an obstacle to a global AIDS vaccine. Viral diversity and host variations in MHC expression both affect vaccine responses. Whether CD8 T cells from HIV-infected donors in 1 part of the world cross-recognize isolates from other regions will provide guidance about whether country-specific vaccines are needed. We compared recognition of HIV_IIIB and representative B′ (Thai B) and recombinant C/B′ virus strains endemic in China by CD8 T cells from 7 HIV-infected North American donors and 4 Chinese donors. IFN-γ production in response to HIV_IIIB or the Chinese viruses was comparable. Although 1.6 ± 0.8% of American donor CD8 T cells produced IFN-γ above the background level in response to IIIB virus, 1.5 ± 0.8% responded to B′ virus, and 1.4 ± 0.7% responded to C/B′ virus. Responses to adherent cells infected with vaccinia viruses expressing B′ and C/B′ virus gag and env were also comparable in magnitude with responses to IIIB virus. Cytolysis of CD4 T cells infected with B′ virus was comparable with lysis of cells infected with IIIB virus, but lysis of the more divergent C/B′ virus was somewhat reduced. T cells, selected for IFN-γ production to IIIB virus, also efficiently lysed cells infected with Chinese viruses. Therefore, cross-clade CD8 T-cell responses to IIIB virus and prevalent Chinese viral strains are common.

Developing an AIDS vaccine has proven challenging because of the difficulty generating neutralizing antibodies to a virus with a highly variable envelope protein. Frustration with developing an antibody-based vaccine, together with the understanding that cellular immunity is important in controlling intracellular pathogens, has led to an emphasis on designing vaccines to induce both T-cell and humoral immunity. T cells, which respond to short peptide sequences in the context of MHC antigens, are able to recognize all viral proteins, including regulatory proteins and enzymes. Although these proteins are less variable than the envelope protein, their sequences also vary significantly between individuals in the same geographic region and even more so between different viral clades. Single point mutations in the 8- to 11-amino acid peptide sequence recognized by the CD8 T-cell receptor can reduce binding affinity and even abrogate T-cell recognition.¹ Information about the extent of cross-clade T-cell reactivity is important for developing immunization strategies for vaccines to be used throughout the world. Although the ultimate test of whether vaccines will have to be custom-made for each region of the world will require clinical studies, a high frequency of cross-clade recognition would make it reasonable to consider testing vaccines expressing proteins from a single clade virus in multiple geographically dispersed sites.

Most studies of antiviral T-cell responses have used assays based on laboratory clade B virus strain peptides or recombinant vaccinia viruses expressing clade B virus antigens and, therefore, provide little information about the extent of cross-clade T-cell recognition in the setting of viral diversity. A few studies using peptides or vaccinia viruses expressing HIV genes from >1 clade in infected humans or vaccinated nonhuman primates and humans have suggested that cross-clade recognition is not uncommon despite the divergence in nucleic acid and protein sequences.^2-11 Durali et al¹² reported frequent cross-reactivity in the CTL response to clade A and B viruses, particularly in recognizing Gag and Nef proteins. Other studies showed that Africans infected with non-clade B virus could mount a vigorous CTL response to >1 HIV clade B protein^13-17 and that CTLs from HIV-2-infected patients cross-react with HIV-1.^18,19 To date, there have been no studies of cross-clade recognition of endemic Chinese and clade B viruses.

The HIV epidemic has spread extensively in China since 1995, with an official estimate of 800,000 people currently infected. Projections suggest that this number may climb to 10 million by the year 2010 if vigorous control measures are not rapidly instituted.²⁰ The Chinese epidemic initially spread from intravenous drug users infected with clade B virus strains in Yunnan Province bordering the Golden Triangle, but soon clade B virus strains were overtaken by clade B′ and C virus infections introduced into southwestern China by drug trafficking from Myanmar, Laos, Thailand, and India.^21-24 A careful national molecular epidemiology survey based on sequencing the env, gag, tat, vif, and nef genes isolated from HIV-infected samples from throughout China by the National Center for AIDS Prevention and Control in collaboration with provincial public health laboratories tracked the spread of the epidemic to all regions of China.^25,26 These studies were facilitated by the establishment of a network of 1400 HIV testing laboratories throughout China from 1996 to 1999, which was built upon the extensive preexisting public health/epidemiology network in China. Although intravenous drug users represent the major source of infection spread in China, transmission through unregulated plasma collection in the mid-1990s and through steadily increasing sexual activity has also become important and has contributed to the genetic diversity of the current epidemic. Eight HIV-1 subtypes (A, B, B′ [Thai B], C, D, E, F, and G) as well as HIV-2 have been isolated in China.^27-29 The most prevalent strains are B′, C, and E. Clade E virus infection has spread by sexual contact and is mostly limited to southeastern China. One of the special features of the HIV epidemic in China is the emergence of an intersubtype C/B′ recombinant virus.^30,31 The recombinant first appeared in Yunnan on the southwest border, soon surpassed the nonrecombinant parental strains, and was transmitted along a drug trafficking road to the northwest border. Preliminary data suggest that the emergent C/B′ virus strain may have developed as a favorable adaptation to the human host. The C/B′ recombinant virus may be more transmissible than the parental strains from which it is derived, yet patients infected with the recombinant virus have lower plasma viral loads and a more benign disease course.³²

Protein homology between the representative Chinese viruses and IIIB virus varies according to the HIV gene product.^30,31 For all proteins except reverse transcriptase, not surprisingly, B′ virus is closer to IIIB virus than C/B′ virus. The amino acid identity of the representative B′ virus strain (RL42) and HXB.2 sequences vary between a low of 72% for gp160 to 93% for the gag polyprotein. Comparing the representative C/B′ virus strain CN54 to IIIB virus, the protein homology varies from 59% identity for gp160 to 82% identity for the gag precursor protein to 92% for reverse transcriptase. Although IIIB virus is not an ideal consensus virus representative of the most prevalent HIV sequences in North America, we previously found by T-cell receptor sequencing and cytokine production assays that CD8 T cells responding to HIV_IIIB overlap substantially with CD8 T cells responding to autologous virus for most stage A HIV-infected subjects, who made up the donors in this study.³³

In this study, we took advantage of recent methods to measure T-cell cytokine production and cytotoxicity against uniform populations of HIV-infected primary cells to quantify the extent of cross-clade reactivity within the CD8 T-cell response to viral antigens on infected primary cells.^3,34 This approach has the unique advantage of avoiding making assumptions about which T-cell antigens might be more important than others, which could conceivably differ across clades, and of measuring responses only to those antigens that are effectively processed and presented on infected primary cells, which closely approximate the in vivo situation, in which levels of presented antigenic peptides may be rate limiting. When we analyzed IFN-γ production and cytotoxicity by CD8 T-cells from North American and Chinese HIV-infected donors, we found no significant differences in responses to clade B (IIIB) virus compared to representative Chinese B′ and C/B′ viruses.

METHODS

Study Population

This work was carried out using thawed PBMCs from 7 stage A HIV-infected subjects living in the vicinity of Boston or Cleveland and 2 healthy volunteers and 4 HIV-infected donors residing in regions of China where HIV is endemic (2 in Xinjiang Province and 2 in Shan Xi Province). The Institutional Review Committees approved the study. Blood samples were taken after obtaining informed consent, and PBMCs were isolated by Ficoll-Paque Plus (Pharmacia, Piscataway, NJ) density centrifugation and cryopreserved using a Gordinier Model 9000 programmed cell freezer (Roseville, MI).

HIV Strains

HIV_IIIB (NIH AIDS Research and Reference Reagent Program) was grown in H9 cells or in autologous PHA-stimulated CD4 T cells as previously described.³⁴ Chinese C/B′ virus was generated by transfecting H9 cells with the molecular clone derived from isolate 97cn54 (CN54), which was selected as the isolate with highest homology (99.6%) to the consensus sequence of type C virus endemic in intravenous drug users in China.³¹ This mosaic virus has 10 recombination sites between B′ and C viruses and is mostly composed of sequences homologous to clade C virus of Indian origin, except for several insertions of B′ virus sequences in the gag-pol coding region, 3′-vpr, all of vpu, the first exons of tat and rev, and the 5′ region of nef. A molecular clone of Chinese B′ virus (RL42) homologous to Thai B′ virus was similarly prepared from a primary virus isolated from an intravenous drug user in Yunnan Province at an early stage of the Chinese epidemic.³⁵

Recombinant Vaccinia Viruses

Recombinant vaccinia viruses in the WR stain backbone expressing no insert (vP1170), lacZ (vSC8), the BH8 isolate of IIIB env (vPE16), and HXB.2 gag (vDK1 and vP1287)^36,37 were obtained from B. Moss or the NIH AIDS Research and Reference Reagent Program. Recombinant vaccinia viruses expressing gag and env genes from the Chinese molecular clones were synthesized, according to a previously described method³⁸ by inserting the genes under the control of the vaccinia 7.5-kb immediate early promoter into the replication-competent Tiantan strain of vaccinia viruses (pSDA1175 vector). Because the env gene contains 2 TTTTTNT sequences, a termination signal for the vaccinia virus early p7.5 promoter, these sequences were mutated before insertion by PCR mutagenesis, without altering the corresponding protein amino acid sequence. Recombinant viruses were selected by plaque assay, and the expression of HIV genes was verified by Western blotting. Viral stocks for all recombinant viruses were produced in HeLa cells and titered by plaque assay on CV-1 cells, with titers between 1 and 3 × 10⁹ pfu/mL. A multiplicity of infection of 5 was used to infect adherent PBMCs overnight. Vaccinia virus was inactivated by ultraviolet irradiation for 10 minutes in medium containing 5 μg/mL psoralen as described previously³⁹ before infected cells were used as stimulator cells.

HIV-Infected Primary CD4 T cells

Uniformly HIV-infected primary CD4⁺ T cells, generated as previously described,⁴⁰ were infected with HIV_IIIB or Chinese C/B′ or B′ virus for use as target cells in cytotoxicity assays and as stimulator cells for IFN-γ production. Autologous CD4⁺ T cells were positively selected with Miltenyi Biotec (Auburn, CA) CD4 beads and cultured for 4 days in medium containing 15% FCS and 120 IU/mL rIL-2 (Chiron Oncology, Emeryville, CA) after activation with 4 μg/mL PHA. Cells (2-3 × 10⁶) were treated with 8 μg/mL polybrene for 2 hours at 37°C, washed, centrifuged with HIV (100-200 ng p24) at 1100 g for 80 minutes, and cultured at 37°C for 36 hours before washing. Five to 7 days later, uninfected cells were removed using Dynal (Oslo, Norway) CD4 beads following the manufacturer's protocol. Uniform HIV infection of the negatively enriched population was confirmed by flow cytometric analysis of p24 expression as described previously using KC57 monoclonal antibody.⁴⁰ Generally, >75% of CD4-depleted cells stain above the background level for intracellular p24. However, the proportion of infected cells, capable of presenting antigen to CD8 T cells, may be higher because T cells can react to only a few peptide-MHC molecules on the cell surface, well below the sensitivity of flow cytometry. Moreover, adding antigenic peptide to CD4-depleted infected stimulator cells does not enhance their cytolysis by peptide-specific CD8 T cells.⁴¹

Intracellular Cytokine Staining

PBMCs were stimulated either with uninfected or uniformly HIV-infected CD4 blasts or with recombinant vaccinia virus-infected adherent cells in RPMI 1640 medium supplemented with 10% FCS at a ratio of 10:1 (PBMCs:stimulator cells). After 12 hours of incubation at 37°C, cultures were treated with 10 μM Brefeldin A for an additional 4 hours. Cells were resuspended in FACS buffer (2% FCS, 0.02% NaN₃ in PBS), stained for cell surface CD8 (Cy5) and CD69 (PE), washed, fixed, permeabilized using the Caltag (Burlingame, CA) Fix and Perm kit, and stained for IFN-γ-FITC (R&D Systems, Minneapolis, MN) as described previously.⁴⁰ CD8 T cells in a tightly gated lymphocyte population were analyzed on a FACScalibur with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ). The percentage of HIV-specific IFN-γ-secreting cells was calculated by subtracting the percentage of IFN-γ-positive cells in simultaneous control cultures stimulated with uninfected CD4 blasts or control vaccinia virus (vSC8)-infected adherent cells. Cells stained with isotype-matched FITC-MsIgG1 (R&D Systems) were used to set the gates.

Selection of HIV-Specific CD8 T Cells

HIV peptide-specific CD8 T cells were generated by culturing HIV-seropositive donor PBMCs with adherent autologous PBMCs preincubated for 1 hour at 37°C with 5 μg/mL immunodominant HIV peptide as previously described.⁴² Recombinant IL-2 (600 IU/mL; Chiron Oncology) was added 2 days later, and the cells were cultured for 10-14 days before use as effector cells in cytotoxicity assays. To isolate IFN-γ-producing cells, 12 hours after stimulation of CD4-depleted PBMCs with HIV_IIIB-infected CD4 T cells as mentioned above, IFN-γ-producing cells were captured with an IFN-γ catch reagent (Miltenyi Biotec) as previously described.³³ The captured cells were cultured in the presence of allogeneic feeder PBMCs and recombinant IL-15 (25 ng/mL; R&D Systems) for 2-3 weeks before use as effector cells in cytotoxicity assays.

Cytotoxicity Assay

Effector cells were prepared as mentioned above using positively selected CD8 T cells obtained using the Miltenyi Biotec Magnetic Separation MiniMACS System following the manufacturer's protocol selected cells were cultured at 5 × 10⁵ cells/mL for 5 days in medium containing 15% FCS, 60 IU/mL rhuIL-2 (Chiron Oncology), and 25 ng/mL IL-15. Uniformly HIV-infected or mock-infected CD4 T cells, produced as described above, were labeled with ⁵¹Cr (3.7 MBq) for 1 hour, washed 3 times in RPMI 1640 medium with 10% FCS, and added (5-10 × 10³ cells in 100 μL) to replicate wells of U bottom microtiter plates before adding effector cells suspended at various effector:target cell (E:T) ratios in an equal volume. The plates were then incubated at 37°C over CO₂ for 4 hours. Supernatants (40 μL) were counted on a Top Count (Packard, Meriden, CT) microplate reader, and percent specific cytotoxicity was calculated from the average cpm as follows: ([average cpm - spontaneous release]/[total release - spontaneous release]) × 100. The spontaneous release for all experiments was within acceptable limits of <20%. HIV-specific cytotoxicity for each virus was calculated as the difference between the specific cytotoxicity against HIV-infected primary CD4 blasts and that against uninfected CD4 T-cell blast controls.

Statistical Analysis

Comparisons in functional responses to different viruses were made by the paired 2-sided Student t test.

RESULTS

Cross-Clade IFN-γ Production in Response to Chinese Viruses in American Donor Samples

To avoid assumptions about which HIV gene products might be important for T-cell recognition of HIV, we used HIV-infected CD4 T cells as stimulator cells in IFN-γ production assays. To quantify the extent of cross-clade recognition, PBMCs from 6 HIV-seropositive North American donors and 2 normal donors were analyzed for IFN-γ production by CD8 T cells in response to autologous CD4 T-cell blasts, which were uninfected or infected with IIIB virus and the representative Chinese B′ and C/B′ viruses. After immunomagnetic removal of uninfected cells with Dynal CD4 beads, p24 staining of infected stimulator cells was uniform and similar in cultures infected with each of the 3 viruses (Fig. 1A). Representative flow cytometry plots are shown in Figure 1B, and a summary of all the data is provided in Table 1. Neither of the 2 normal donor samples had detectable IFN-γ-producing CD8 T cells above the background level in response to any of the 3 HIV strains. In the 6 HIV-seropositive samples, there was some background response to CD4 blasts that were not exogenously infected; a mean of 0.21% (range, 0.11-0.32%) of CD8 T cells produced IFN-γ without added virus. In another study,³³ assays were performed using uninfected CD4 blasts cultured in the presence of a cocktail of ritonavir, saquinavir, and zidovudine, and the frequency of IFN-γ-producing CD8 T cells was <0.10%. Therefore, some of the background response can be attributed to small amounts of autologous virus in activated CD4 T-cell cultures not treated with antiviral drugs. Nonetheless, after subtracting the background response to uninfected blasts, substantially more IFN-γ-producing cells were present in the cultures stimulated with autologous cells infected with IIIB virus; 1.63 ± 0.82% of CD8 T cells produced IFN-γ in response to IIIB virus-infected autologous cells. The response to stimulator cells infected with the Chinese virus isolates was almost as great: 1.52 ± 0.82% in response to the representative B′ virus and 1.35 ± 0.69% in response to the representative C/B′ virus. The difference in rates of response to B′ and C/B′ viruses compared with IIIB virus was not statistically significant by the paired 2-sided Student t test (P = 0.79 and P = 0.37, respectively).

Table 1

Figure 1

Cross-Clade Reactivity of CD8 T Cells to Macrophages Infected With Recombinant Vaccinia Viruses Expressing Clade B, B′, and C/B′ Genes

To verify further the high degree of cross-clade reactivity in the CD8 T-cell response, for 1 donor (606) we also measured IFN-γ production in response to adherent PBMCs infected with recombinant vaccinia viruses expressing env and gag genes of the 3 viruses (Fig. 2). Because adherent cells in PBMCs are mostly of macrophage lineage and macrophages represent a major cell type responsible for viral transmission, this assay tests whether there is a high degree of cross-clade recognition of the cells that needs to be eliminated upon initial viral exposure; 0.41% of donor 606 CD8 T cells produced IFN-γ in response to adherent cells expressing IIIB virus gag and 0.18% responded to IIIB virus env, while only 0.05% responded to the vaccinia virus lacZ control. The responses to adherent cells infected with vaccinia viruses expressing B′ and C/B′ virus gag and env were comparable in magnitude with the responses to IIIB virus. Surprisingly the response to C/B′ virus gag was greater than the response to IIIB virus gag, while the response to B′ virus gp120 was greater than the response to IIIB virus gp120. This finding was reproducible. The reasons for this are unclear, but they could be related to differences in antigen presentation or to linear viral sequences of epitopes.

Figure 2

Cross-Clade Reactivity in Cytotoxicity Assays

To determine whether the high degree of cross-clade reactivity detected for IFN-γ production also applied for cytotoxic function, which may require more potent T-cell receptor stimulation than IFN-γ production,⁴³ T-cell lines were generated from 3 HIV-seropositive donors and tested for their ability to lyse primary CD4 T cells infected with IIIB virus and the two Chinese viruses (Fig. 3). In all 3 samples, there was no background lysis of uninfected autologous CD4 blasts. Lysis of CD4 T cells infected with B′ virus was comparable with lysis of CD4 T cells infected with IIIB virus. Lysis of the more divergent C/B′ virus was somewhat reduced compared with lysis of IIIB virus. At an E:T ratio of 25:1, specific lysis of IIIB virus-infected autologous CD4 cells was 34 ± 5%, while lysis of B′ virus-infected cells was 31 ± 2% and that of C/B′ virus-infected cells was 24 ± 6%. Neither of these differences was statistically significant, but the sample size was small (IIIB virus vs. B′ virus, P = 0.60; IIIB virus vs. C/B′ virus, P = 0.13).

Figure 3

Variable Cross-Clade Recognition by Antigenic Peptide-Specific T-Cell Lines From 1 Donor

For 1 donor (606), we also examined cross-reactivity of T-cell lines stimulated with autologous B lymphoblastoid cells incubated with 19- to 22-amino acid HXB.2 and SF2 HIV peptides containing epitopes from gag, env, and nef recognized by this donor. Mapping of the fine specificity of the epitopes has not been done. The peptide-stimulated lines were then tested for cytolysis of autologous CD4 T cells infected with HIV_IIIB or the representative strains of virus from China (Fig. 4). Although lysis of autologous IIIB virus-infected CD4 T cells for each of the lines was low (4-18% at an E:T ratio of 10:1), there was no background lysis of uninfected CD4 T cells. These cell lines were able to lyse Chinese B′ virus-infected CD4 T cells to a similar extent as IIIB virus, but the lysis of C/B′ virus-infected targets was consistently less and was not detected at all using gp41 peptide-stimulated cells as effector cells. The gag peptide was identical to the sequence in IIIB virus and differed from the sequence in B′ and C/B′ viruses by a single Ala to Pro substitution (Fig. 4). The gp41 and nef peptides used for stimulation, although from clade B viruses, differed from the IIIB virus sequence at 1 residue. B′ and C/B′ viruses had identical sequences in the region of the nef peptide and differed from the stimulating peptide at 2 residues. Although B′ and C/B′ viruses had identical peptide sequences for gag and nef, C/B′ virus-infected cells were consistently less well lysed than B′ virus-infected cells. This may be because the cell lines recognize other B′ virus epitopes not presented by C/B′ virus or because antigen presentation by C/B′ virus-infected cells to T cells was less efficient than by B′ virus-infected cells for reasons other than differences in the antigenic sequence. It is not surprising that the env peptide-stimulated cell line did not recognize cells infected with C/B′ virus, because the env peptide sequence of C/B′ virus differs at 6 of 20 residues from the stimulating peptide.

Figure 4

Cross-Clade Cytotoxicity by CD8 T Cells That Produce IFN-γ in Response to IIIB Virus

Because the frequency of HIV-specific CD8 T cells in these samples was low, we also examined cross-clade cytotoxicity by highly potent HIV-specific CD8 T-cell lines generated from 2 donors (307 and 606) by capturing cells producing IFN-γ in response to HIV_IIIB-infected autologous CD4 T cells. These cells, selected using a previously described method,³³ were expanded until they reached sufficient numbers to perform the assay. Even at E:T ratios of 1:1, these cell lines lysed more than one half of the IIIB virus-infected targets and had no background response to uninfected autologous CD4 T-cell blasts (Fig. 5). For donor 307, lysis of both B′ virus- and C/B′ virus-infected cells was similar and somewhat more than half of what it was against IIIB virus-infected CD4 T cells. For donor 606, B′ virus-infected cells were lysed about as well as IIIB virus-infected cells, but lysis of C/B′ virus-infected cells was about half as great. These results for sample 606 are consistent with the findings in Figures 3 and 4 using either bulk T cell lines or peptide-specific lines generated from this donor as effector cells.

Figure 5

CD8 T Cells From HIV-Infected Chinese Donors Efficiently Recognize HIV_IIIB

All of the above studies looked at recognition of representative Chinese viruses by CD8 T cells from North American donors. We next looked at whether T cells from Chinese donors respond as strongly to gag polypeptide encoded by HIV_IIIB as to gag derived from Chinese isolates. IFN-γ production in response to adherent stimulator PBMCs infected with recombinant vaccinia viruses expressing the various gag genes was studied in 2 donors from Xinjiang Province who were infected with C/B′ virus and 2 donors from Shan Xi Province who were infected with B′ virus (Fig. 6). Although the Xinjiang donors tended to have a higher proportion of T cells responding to C/B′ virus gag than to B′ or IIIB virus gag and the Shan Xi donors responded better to B′ virus gag than to C/B′ virus gag, the differences in recognition were small and not significant. Moreover, the mean response above the background level to HIV_IIIB gag (1.2 ± 0.3% of CD8 T cells) was not significantly different from the mean response to B′ virus gag (1.2 ± 0.3%) or C/B′ gag (1.2 ± 0.6%). Therefore, the Chinese donor CD8 T cells, like those from the HIV-infected North American donors, had a high proportion of cross-reactive cells.

Figure 6

DISCUSSION

In this study, we compared the ability of circulating T cells from 7 asymptomatic North American HIV-infected donors and 4 Chinese donors to produce IFN-γ and/or lyse autologous primary cells infected with a laboratory-adapted clade B virus strain (IIIB) and 2 representative strain viruses from endemic regions in China. We found a substantial amount of cross-clade CD8 T-cell functional responses to the Chinese endemic viruses among North American donors and to HIV_IIIB among the Chinese donors, with no statistically significant differences. IFN-γ production or cytotoxicity by CD8 T cells in response to HIV_IIIB and to the Chinese representative strains was of similar magnitude when assayed using virus-infected CD4 T cells, recombinant vaccinia virus-infected macrophages, or antigenic peptide-loaded B-LCLs as target cells. These results suggest that many of the T-cell responses to relatively conserved clade B virus epitopes are also conserved more broadly across clades. This may be because the epitopes recognized during chronic infection have already persisted in the face of CTL escape mutation^44-47 and represent relatively well-conserved sequences that cannot readily be mutated without interfering with viral fitness. The implication is that at least the T-cell arm of a vaccine may not need to be customized for different regions of the world. This study using Chinese viral isolates confirms results obtained using other techniques (peptide-armed and vaccinia virus-infected antigen-presenting cells) to examine samples from other parts of the world, particularly Africa.

Using virus-infected primary cells as targets provides a physiologically relevant way to measure cross-clade T-cell responses. This approach, pioneered by Ferrari et al,³ obviates the need to prepare customized recombinant vaccinia viruses or pools of peptides to study antiviral responses to primary viral isolates. Moreover, vaccinia viruses, which suppress host-cell protein translation, and peptide-loaded targets provide an excess of viral antigens, which may not reflect the restricted amounts of HIV antigen available for processing and presentation in naturally infected cells. Using infected cells as targets also allows measurement of all the possible responses to viral antigens, without presupposing that some viral proteins are more important than others are. The dominant recognized antigens vary from 1 HIV-infected individual to another, even when the HLA background is shared, probably because of prior differences in antigen exposure to other cross-reacting infectious agents.^48-50

Similar methods were recently used to compare the magnitude of IFN-γ production by CD8 T cells from HIV-infected donors in response to autologous virus and IIIB virus.³³ In CDC stage A donors, but not symptomatic patients, there is a similar magnitude CD8 T-cell response to IIIB virus and to autologous virus. Moreover, the T cells recognizing autologous virus cross-react with those recognizing IIIB virus and vice versa. Therefore, in this study, measuring responses to HIV_IIIB was a reasonable surrogate for measuring CD8 T-cell responses to autologous clade B virus, because all the donors studied had stage A disease.

One of the Chinese viruses is highly related to Thai B′ clade virus (RL42), and the other is a recombinant virus (CN54) containing sequences derived from Thai B′ virus and Indian clade C virus. Recombination analysis of C/B′ virus suggests 10 recombination sites, although most of the gag, pol, and env sequences of the recombinant virus are most closely related to clade C virus.³¹ C/B′ virus shares ∼94% nucleotide homology to a consensus clade C virus sequence of gag, pol, and env and 78-94% homology to the regulatory genes. Compared with consensus clade B virus, the C/B′ virus sequence shares homologies of 90% for gag, 92% for pol, 85% for env, and 88% for nef. Compared with the Chinese B′ virus isolate used in this study, the nucleotide homologies are 92% for gag, 91% for pol, 83% for env, and 86% for nef. Although a single amino acid change in a T-cell epitope can abrogate recognition,¹ apparently this degree of viral diversity between the isolates studied here had little impact on overall CD8 T-cell recognition. However, not surprisingly, variations in sequence did affect the recognition across clades of T-cell lines from 1 donor that were selected to be specific for particular epitopes. This was particularly true for the most variable env sequence. The difference between high levels of cross-clade reactivity using HIV or recombinant vaccinia virus-infected targets compared with more variable cross-clade recognition when particular epitopic responses are studied argues against designing vaccines based on incorporation of a small number of peptidic epitopes. Overall, our data suggest that at least in the chronic phase of disease much of the CD8 T-cell response is focused on relatively well-conserved sequences.

Our results and previous studies that examined cross-clade recognition of HIV^{3,5-7,10,11,13,15-17,51,52} suggest that HIV vaccines designed to induce T-cell reactivity may not need to be tailor-made for different clades or be composed of cocktails of constructs derived from viruses endemic to the region where the vaccine is being used. Moreover, several vaccines have induced some degree of cross-clade reactive T-cell reactivity.^4,8,53 Clade-specific vaccines are more likely to be required of vaccine components used to induce neutralizing antibodies because of the high sequence variability of HIV gp160. However, some neutralizing determinants elicit cross-clade responses, and some antibodies that are clade specific have broad cross-clade activity when used in combination.^54-66 However, only clinical testing with an effective vaccine will resolve the question of whether a single vaccine can generate protective immunity across clades.

The relevance of cross-clade CD8 T-cell reactivity to the development of an effective AIDS vaccine remains uncertain. Until an effective vaccine becomes available, we will not know what sort of immune response will be able to provide effective protection. The CD8 T-cell response may not be critical, although many researchers suspect that an effective vaccine will probably need to elicit both humoral and cellular immunity. In international settings, induction of effective CD8 T-cell responses will depend not only on having relevant epitopes in the vaccine immunogen, but also on host factors such as genetic variations in immune response genes, nutritional status, and the prevalence of intracurrent infections.

ACKNOWLEDGMENTS

The authors thank Christoph Lange and Michael Lederman (Cleveland Center for AIDS Research) for providing samples for 1 of the donors in this study, Jianqing Xu for helpful suggestions, and Chiron Oncology for recombinant human IL-2.