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Sequential priming and boosting with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes

Xu, Jianqing; Ren, Li; Huang, Xianggang; Qiu, Chao; Liu, Yong; Liu, Ying; Shao, Yiming

doi: 10.1097/QAD.0b013e328010ad0c
Basic Science

Background: The effort to develop an effective preventive vaccine against HIV-1 infection is challenged by the wide genetic diversity of HIV-1 among different isolates.

Objectives: To explore a new vaccination strategy by using heterologous HIV immunogens derived from different clades for sequential priming and boosting.

Methods: HIV Env and Gag immunogens derived from Thailand B (B′), C/B′ recombinant and A/E recombinant were selected as these three clades account for 29%, 45% and 15% of HIV-1 prevalence in China, respectively. Three humanized fusion genes of env and gag derived from those three clades were synthesized and inserted into DNA and recombinant Tiantan vaccinia vectors as model vaccines. C57BL/6 and Balb/c mice were used as animal model. Peptides spanning the entire Env and Gag were used as stimuli and Elispot assay was used to assess the T cell immunity.

Results: Sequential priming and boosting was observed with heterologous HIV immunogens predominantly stimulated T cell immunity against conserved epitopes, whereas a single vaccine derived from one clade or the mixture of multiple vaccines from different clades primarily raised T cells against less conservative or non-conservative epitopes.

Conclusions: This is the first demonstration of a practical strategy to raise immune responses against conserved epitopes. This strategy has important implications for vaccine development against HIV and other pathogens that have high genetic diversity, such as influenza.

From the State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention (NCAIDS), Beijing, China.

Received 8 June, 2006

Revised 8 August, 2006

Accepted 8 September, 2006

Correspondence to Dr Jianqing Xu or Dr Yiming Shao, Room 713, National Center for AIDS/STD Control and Prevention (NCAIDS), China CDC, 27 Nanwei Rd, Xuanwu District, Beijing, 100050, China. E-mails:,

Jianqing Xu and Li Ren equally contributed to this project.

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An effective vaccine against HIV-1 remains elusive despite more than two decades of intensive global effort. This effort has been hampered by the wide genetic diversity in HIV-1 [1]. Currently, HIV-1 group M constitutes the majority of HIV-1 isolates and dominates the global AIDS epidemic. Group M can be divided into at least nine clades, designated A, B, C, D, F, G, H, J and K, and a number of different circulating recombinant forms (CRF). The amino acid sequence variation in envelope proteins within group M ranges from 3 to 23% among isolates of the same clade, and from 25 to 35% among different clades. Since it is highly unlikely that a vaccinated individual will be infected in the future by the same isolate or laboratory strain from which the vaccine has been derived, the cross-reactive immune responses will be vital for containing this subsequent HIV-1 infection. Therefore, an effective HIV preventive vaccine must be capable of mounting a high level of cross-reactive immune responses that can react to different isolates within the same clade and even to different clades.

We hypothesized that cross-clade HIV-specific immune responses (cross-reactive to different clades) could be raised and enhanced by sequential priming and boosting with heterologous HIV immunogens. Sequential priming and boosting with heterologous HIV immunogens (hereafter refers as to ‘heterologous vaccination’) is defined as a vaccination strategy using different vaccines, derived from different clades, for sequential priming and boosting. For example, in a vaccination scenario containing three sequential inoculations, vaccines derived from an isolate of E and B clade can be used in the first and the second inoculation, respectively, and then a vaccine derived from the C clade can be used for the final inoculation. It was considered that, compared with conventional vaccination strategies, the advantage of heterologous vaccination would be that only cross-clade HIV-specific immune responses targeting at conserved epitopes would be sequentially enhanced, thereby dominating the total HIV-specific immune responses; the non-cross-reactive immune responses would be established but not boosted. Interestingly, it has been shown that cross-reactive T cells become dominant after sequential infections by different viruses and are maintained at high frequencies in the memory pool while non-cross-reactive T cells are selectively lost [2]. In addition, mismatch Env between immunogens and challenge viruses might result in the expansion of cross-reactive immune responses at the early stage of infection and thereby lead to a better control of viral replication and an improved protection from CD4 T cell loss [3]. These observations suggest that the immune system has evolved a mechanism to develop cross-reactive T cells during viral infections, which may be important to allow a minimal effort to achieve a maximal effect.

HIV-1 became prevalent in the early 1990s in Yunnan province in China with the entry of HIV-1 B′ subtype from Thailand, which was followed by HIV-1 subtype C entering into Yunnan province from India. Subtypes B′ and C recombined into a C/B′ recombinant in Yunnan province and further spread to northwestern China. An A/E recombinant entered into southern China in the mid 1990s, and became prevalent in southern and southeastern China later. According to a nationwide molecular epidemiology study (1996–2003), the most prevalent strains in China are C/B′ recombinant form (accounting for 45% prevalence), Thailand B (B′) (accounting for 29% prevalence) and A/E recombinant form (accounting for 15% prevalence, originally designated as E) (unpublished data).

This study tests the sequential cross-clade vaccination strategy using vaccines derived from a C/B′ recombinant strain, CN54 (of which the entire env and approximately 70% gag were derived from C clade [4]), a B′ strain, RL42, and an A/E recombinant strain, AE2F. CN54, RL42 and AE2F strains were isolated during the very early prevalence studies of each subtype [4–6] and, therefore, might be considered as ancestor-like sequences in China, though sequence data to establish this concept are still unavailable.

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Animals and vaccination

All DNA vaccines and vaccinia-vectored vaccines used in this study were previously constructed with confirmed expression efficacies by p24 intracellular staining and Western blot assay. Only gag and env genes from each clade were included in vaccine constructs. The vaccination strategies compared in the mice model were conventional with a single vaccine (referred to as single in the figures) or with a mixture of multiple vaccines (referred to as mixture) or the heterologous strategy (referred to as heterologous cross-clade).

All animals experiments were reviewed and approved by the Institutional Animal Care and Use Committee at China CDC animal facility and were performed in accordance with relevant guidelines and regulations. BALB/c and C57BL/6 mice (6–8 weeks old, 18–22 g, male or female) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College. All experiments were carried out in a specific pathogen-free animal facility and all animals were anesthetized using 1% barbital sodium before immunization. Four groups of nine BALB/c and four groups of nine C57BL/6 mice were randomly immunized as scheduled. The vaccines used were a C/B′ recombinant strain (CN54), a B′ strain (RL42) and an A/E recombinant strain (AE2F); these are referred to in this study as C, B′ and E vaccines, respectively. Group 1 as control group was primed with mock DNA vector pDRVI-SV1.0 derived from pVRC2000 and boosted with mock Tiantan vaccinia. All other groups received 100 μg DNA vaccines encoding Gag and Env at weeks 0 and 2 and 1 × 107 plate-forming units Tiantan vaccinia-vectored vaccines at week 5. Group 2 received the single B′ clade-derived vaccine at every time; group 3 received sequential cross-clade vaccination (E, B′, then C at weeks 0, 2, and 5, respectively); group 4 was immunized with the mixture of multiple vaccines (E + B′ + C at each time). Mice were then sacrificed at week 7 for mapping peptide pools and at week 8 for mapping single peptides. All experiments were repeated in a second round. Overall, 144 mice were used: 72 C57BL and 72 Balb/c, divided into duplicate treatment groups 1–4, each group containing nine mice. In all, within each treatment group, eight mice were used for mapping peptide pools and 10 for mapping single peptides.

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Formulation of peptide pools and Elispot assay

Both consensus B and consensus C 15-mer peptide complete sets spanning the entire Gag and Env proteins were included to formulate peptide pools. One Env peptide pool comprised six peptides: three from consensus B and three from consensus C. The one Gag peptide pool comprised 10 peptides: five from consensus B and five from consensus C. This fitted into a matrix on one 96-well plate. The two-dimensional location on the plate identified the peptide pools; for example, F10 refers to the peptide pool formulated by consensus B and C Gag peptide 36–40. Two weeks after the final inoculation of rTTV vaccines, mice were sacrificed and spleocytes were harvested. The interferon-γ Elispot kit (BD Biosciences, San Diego, California, USA) was used to determine vaccine-elicited IFN-γ Elispot responses in both Balb/c and C57BL/6 mice. The 96-well plates were coated with purified anti-mouse IFN-γ monoclonal antibodies at 5 μg/ml in 100 μl per well, and incubated at 4°C overnight; the wells were washed three times with 200 μl/well R10 complete culture medium (RPMI-1640 containing 10% fetus bovine serum and 1% penicillin–streptomycin–L-glutamine), and blocked with 200 μl/well R10 complete culture medium at room temperature for 2 h. Mice splenocytes were separated and red blood cells (RBC) were lysed by RBC Lysis Buffer (139.6 mmol/l ammonium chloride, 16.96 mmol/l Tris, pH 7.2). Cells were then washed twice with R10 and resuspended in R10 complete culture medium. After counting the splenocytes, they were adjusted to 3 × 106 cells/ml and plated into precoated 96-well Elispot plate at 100 μl/ well with addition of 4 μl peptide or peptide pools (at 1 μg/μl in stock solution), the final concentration for each peptide is 4 μg/ml. The plates were incubated for 24 h at 37°C under 5% CO2. After incubation, the plates were developed according to the manufacturer's instructions. Finally, the plates were air-dried and the resulting spots were counted with Immunospot Reader (CTL, Cleveland, Ohio, USA). Peptide-specific IFN-γ Elispot responses were considered as positive only when the response was four-fold above the negative control with no peptide stimulation and the spot-forming cells (SFCs) were > 50 sfc/106 splenocytes. To determine dominant epitopes, all T cell immune responses were converted to an average percentage of T cell responses directing at the corresponding peptide in the total T cell immune responses against both consensus B and C peptides for each group: the sum of against all peptide would be 100%.

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Peptide pool-specific T cell responses with heterologous vaccination

Conventional strategies, including vaccination with a single vaccine and with a mixture of multiple vaccines, were included for comparison, and the empty DNA and Tiantan vaccinia vectors were used as control. The vaccination strategy is shown in Fig. 1a and the peptide pools formulated in Fig. 1b.

Fig. 1

Fig. 1

Fig. 1

Fig. 1

In the four C57BL/6 mice sacrificed at week 7 from each group, the group 1 mice (mock control) did not achieve a stimulation that was greater than four-fold over the negative control (no peptides). By comparison, heterologous vaccination predominantly stimulated T cell immunity against F1, C4, D4, E10 and F10 peptide pools, much less to A1 and D9 pools and only marginally to B12. Immunization with a single vaccine or with the mixture of multiple vaccines markedly activated T cell responses against A1, D9 and B12 peptide pools, much less to F1, C4, D4, E10 and F10 pools (Fig. 1c, left; the original image figure is available from the authors). Interestingly, all vaccination strategies mounted a comparable magnitude of T cell immunity against the most preferential peptide pool, which varied among different strategies (Fig. 1c). Indeed, the total T cell responses against all peptide pools from these three immunization strategies were similar (Fig. 1d); however, since HIV-1 clade E-derived peptides were not included in the quantification of T cell immunity, the current settings may underestimate the differences in immunity raised by different strategies.

To exclude bias caused by genetic background, T cell immunity raised by the different strategies was also examined in Balb/c mice (Fig. 1c, right; the original image figure is available from the authors). Vaccination with a single vaccine or with the mixture of multiple vaccines predominantly elicited T cell immunity against H10 peptide pool and profoundly less to all other peptide pools. While capable of raising marked T cell response against H10 pool, heterologous vaccination also significantly induced T cell immunity against peptide pool F1, F6 and G4.

Overall, heterologous vaccination elicited different patterns of peptide pool-specific T cell responses from single vaccine or a mixture of multiple vaccines both in C57BL and in Balb/c mice. Repeated experiments showed identical patterns of peptide pool-specific T cell responses.

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Individual peptide-specific T cell responses with heterologous vaccination

The individual peptides from the five predominant peptide pools in C57BL mice were mapped, including A1, F1, D9, F10 and B12. Since the total T cell responses against all peptide pools from the three immunization strategies were comparable, the percentages of T cell responses against each peptide in total T cell immunity was calculated to determine the dominant individual peptides. Five immunized C57BL mice from each group were sacrificed for mapping single antigenic peptides. Heterologous vaccination predominantly stimulated T cell responses symmetrically targeting at consensus B and C peptides Env 16, Gag 36 and Gag 37, whereas conventional strategies preferentially activated T cell responses targeting Env 2, Env 3 for vaccination with a single clade B′-derived vaccine alone and Env 202, Env 203 and Gag 92 from consensus B for vaccination either with a single clade B′-derived vaccine or the mixture of multiple vaccines (Fig. 2a). This pattern was repeated in the duplicate experiments.

Fig. 2

Fig. 2

Similar results were observed using Balb/c mice (Fig. 2b). Vaccination with either a single clade B′-derived vaccine or the mixture of B′, C and E vaccines primarily elicited T cell responses targeting Gag 49 of both consensus B and C clades and Gag 50 of consensus B clade, which indicated that the epitope residing within those peptides is highly immunogenic and one amino acid replacement of E in consensus B Gag 49 by D in consensus C Gag 49 does not prevent recognition of consensus C Gag 49 though it is decreased. In contrast, T cell responses against Gag 49 and Gag 50 were much less profound when heterologous vaccination was employed. Instead, T cell responses symmetrically targeting Env 16, Env 17 and Env 137 of both consensus B and C clades, at Env 138 of consensus B clade and at Env 93 and Env 136 of consensus C clade were dramatically improved by heterologous vaccination. This pattern was repeated in the duplicate experiments.

Interestingly, vaccination with the mixture of multiple vaccines targeted more consensus C peptides and thereby stimulated broader T cell immune responses than that seen with a single clade B′-derived vaccine in both C57BL/6 and Balb/c mice, whereas heterologous vaccination mounted more balanced and symmetric T cell immune responses against consensus B and C than seen with conventional vaccination strategies (Fig. 2a,b).

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T cell immune responses targeting conserved consensus epitopes with heterologous vaccination

To decipher why heterologous vaccination stimulates predominant epitopes that are distinct from those in conventional strategies, predominant T cell epitopes were mapped on the sequence alignment of the three vaccine immunogens (Fig. 3). Eight dominant T cell epitopes were identified within Gag and Env in Balb/c and C57BL mice, including three residing in Gag and five in Env. Interestingly, sequential heterologous vaccination predominantly stimulated T cell immune responses against three epitopes, one located at p24 at amino acids 16–23 and recognized by C57BL mice (Fig. 3a), the other two resided at gp160 at amino acids 65–75 and recognized by both C57BL and Balb/c mice and at gp160 at amino acids 545–553 recognized by Balb/c mice (Fig. 3b); All those epitopes were either fully conservative among all three vaccine immunogens or conservative in both priming and boosting immunogens. However, heterologous vaccination stimulated smaller T cell responses against the less conservative epitopes, such as epitopes at p24 amino acids 65–75 and gp160 amino acids 816–826, and it failed to elicit significant T cell immune responses against non-conservative epitopes at p24 amino acids 365–384 and gp160 amino acids 9–19. Overall, the intensity of T cell immune responses stimulated by heterologous vaccination decreased in parallel with the decreased level of sequence consistency among immunogen epitopes.

Fig. 3

Fig. 3

Fig. 3

Fig. 3

In contrast, vaccination with either a single clade B′-derived vaccine or the mixture of multiple vaccines was unable to stimulate a substantial T cell immune response against those fully conservative epitopes; instead, these strategies activated predominant T cell immunity against less conservative or non-conservative epitopes, such as epitopes residing in p24 amino acids 65–75 and 365–384, gp160 amino acids 9–19 and 816–826. One epitope, consensus C Env 93, does not follow this rule. Though this epitope is non-conservative in its amino acid sequences, heterologous vaccination primed higher T cell responses than conventional strategies. We speculate that either this epitope mainly presents in C vaccine (boosting vaccine) or there exist conservative immune epitopes (amino acids sequences may vary but contain conservative antigen specificity) at these sites, just as observed in previous report [7].

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Strategies to raise immune responses preferentially targeting conservative immune epitopes remain to be explored though it has been recognized that an effective HIV-1 vaccine should be capable of eliciting significant cross-reactive immune responses. Early studies using peptides derived from variable region of gp120 have demonstrated that CD8 T cells primed by one peptide are able to recognize or be boosted by homologous determinants containing variant sequence from distinct isolates [7,8]. These studies suggested that it is feasible to prime with one vaccine and to boost with another for raising cross-reactive T cells. Indeed, a recent report on HIV-1 superinfection showed that only one out of nine conserved epitopes developed significant CD8 T cell response (> 1000 SFCs/106 peripheral blood mononuclear cells) during HIV-1 virus A infection whereas an additional six conserved epitopes developed significant CD8 T cells only after infection by the second virus (virus B) [9]. This finding emphasizes the importance of the immune system ‘seeing’ different sequences to develop cross-reactive T cells against conserved epitopes. However, the sequential HIV-1 infection by virus A and B may have severely damaged the immune system as the developed cross-reactive T cell immune responses finally failed to contain HIV-1 replication. Cross-clade HIV-specific T cell immune responses have been observed in Nairobi sex workers who remained seronegative despite frequent exposure to HIV [10].

It has been shown that both the magnitude [11–13] and the breadth [14–16] of immune responses significantly influence the control of viruses. Therefore, current HIV vaccine design strategies are mainly directed at increasing the magnitude by a prime-boost strategy and the breadth by including more HIV genes from the same clade or even from different clades. However, there is no practical strategy to enhance cross-reactive immune responses against conservative epitopes. Here we demonstrated a new strategy, we call heterologous vaccination, that is able to stimulate cross-reactive T cell immune responses predominantly against conservative epitopes; the magnitude of T cell immune responses increase in parallel with the increase in epitope sequential consistency among immunogens. Unlike peptide-based vaccine [7,8], in our strategy the host immune system itself will ‘see’ different viral sequences and determine the conserved epitopes and then optimize the immune responses individually. This new strategy may be crucial for future development of an effective HIV-1 vaccine and for development of vaccines against other pathogens that show such genetic diversity, for example hepatitis C and influenza viruses. Though we have not tested this in current experiments, theoretically B cell immune responses targeting at conservative B cell epitope should be similarly stimulated and enhanced by the sequential heterologous vaccination strategy.

We have shown that the heterologous vaccination strategy preferentially enhances immune responses against the most conserved epitopes among various HIV clades, less to epitopes that are less conserved and much less to non-conservative epitopes (Fig. 4). The strategy differs from that tested in mice or primate models in which multiple envelopes from different clades were used in combination for vaccination [17–18]. In these models, more immunogenic epitopes but not more conservative epitopes mounted higher T cell immune responses. Actually, our data show that vaccination with the mixture of multiple vaccines stimulated broader T cell immunity than that with a single vaccine, indicating that this strategy may be more effective in areas where there are multiple HIV-1 clades circulating. However, it did fail to mount significant T cell immunity targeting at conserved epitopes.

Fig. 4

Fig. 4

To determine whether T cell epitopes raising predominant T cell immune responses in heterologous vaccination are also conserved among other primary isolates, the conservation of one epitope, SPRTLNAW at p24 amino acids 16–23, was examined among different HIV-1 clades in the sequences from Los Alamos database. All primary isolate sequences for CRF07_BC and CRF08_BC were selected and randomly selected sequences for B and C clades (approximately every 10 sequences) and for A/E recombinants (approximately every four sequences). Sequence alignments showed that the epitope SPRTLNAW is highly conserved, appearing in 229 out of 249 sequences (92%) in different clades: 81 out of 93 for B clade (87%), 72 out of 73 for B/C recombinants (98.6%), 61 out of 67 for C clade (91%) and 15 out of 16 for A/E recombinant (93.7%). The biological relevancy of this conservative epitope was examined using the Los Alamos Immune Database, which showed that it is a B*57-restricted epitope. B*57 is an allele closely linked to slow progression during HIV natural infection [19–21]. Importantly, a previous report [22] demonstrated that the capacity to restrain HIV replication for B*57-positive individuals relied on whether the T cell immunity focused on the conservative epitopes; long-term non-progressors mounted highly focused immune responses against conserved epitopes whereas progressors were less focused. This may illustrate the importance of stimulating immune responses directing at conserved immune determinant.

Interestingly, our data showed that the order for conservative epitopes presenting in heterologous vaccination does influence the magnitude of its specific T cell immunity. The conservative epitope presenting in both DNA priming and vaccinia boosting, such as the epitope residing in gp160 amino acids 545–553, elicited much stronger T cell immunity than those presenting twice in DNA vaccinations, such as epitopes locating at p24 amino acids 65–75 and gp160 amino acids 816–826. These data indicate that a strong boosting does but not the second DNA immunization is critical for effective enhancement of the T cell immunity raised in the previous vaccination. Therefore, we speculate that we will observe a different pattern of dominant T cell epitopes, though all are conserved, by changing the order of vaccines for heterologous vaccination. Furthermore, by introducing a strong boosting (such as administrating recombinant adenoviral vectored vaccine) at the second inoculation, we may overcome the drawback of our current vaccination modality and further increase the T cell immunity against the most conservative epitopes. These hypotheses should be explored further in future experiments with larger groups of mice. In addition, this modality should be tested in a primate model with strong boosts to increase the immunogenicity.

The limited availability of peptide sets gave rise to several limitations in this study. The first is that immune responses were assessed using a consensus clade B and clade C peptide set rather than peptide sets corresponding to the vaccine sequences used. In the light of the sequence differences between B′ and C sequences used for vaccination and the clade B and C consensus sequences (the pairwise similarities are 95% between B′ and consensus B Gag, 91% between B′ and consensus B Env, 91% between our C and consensus C Gag, and 87% between our C and consensus C Env), the consensus peptide set will preferentially identify T cell responses that are cross-reactive between the sequences used for vaccination and the peptide sequences, thereby biasing the detection of responses towards conserved areas of HIV-1; In other words, we may have underestimated the differences in immunity primed by different immunization strategies. The second limitation is that clade E-derived peptides were not included for assessing immune responses yet clade E-derived vaccine was included both in heterologous vaccination and in vaccination with the mixture of multiple vaccines; consequently, we might also have underestimated the immune responses specific for clade E sequences. Overall, our current results may have significantly underestimated the immune responses raised by different immunization strategies, particularly by the use of a mixture of multiple vaccines; this would emphasize the breadth and magnitude of immunity primed by this strategy.

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We would like to acknowledge Danli Duan, Yanming Wan, Lianxin Liu and Hong Peng for their kind technical support.

Sponsorship: This research was supported by project 30571709, National Natural Science Foundation of China, and by 973 project 2005CB522903, the China Ministry of Science and Technology, China. HIV-1 consensus B and C Gag and Env (15-mers) peptides (complete sets) were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.

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HIV; vaccine; T cell immunity; conserved epitope; heterologous immunogen

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