A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study

Vaccine 29 (2011) 2918–2932

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A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study Flavia Ferrantelli a , Maria Teresa Maggiorella a , Ilaria Schiavoni a , Leonardo Sernicola a , Erika Olivieri a , Stefania Farcomeni a , Maria Rosaria Pavone-Cossut a , Sonia Moretti a , Roberto Belli a , Barbara Collacchi a , Indresh K. Srivastava b , Fausto Titti a , Aurelio Cafaro a , Susan W. Barnett b , Barbara Ensoli a,∗ a b

National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome, Italy Novartis Vaccines and Diagnostics, Inc., 350 Massachusetts Ave, Cambridge, MA 02139, USA

a r t i c l e

i n f o

Article history: Received 3 May 2010 Received in revised form 31 January 2011 Accepted 5 February 2011 Available online 21 February 2011 Keywords: HIV/AIDS vaccine Neutralizing antibodies Mucosal R5 SHIV challenge

a b s t r a c t HIV native Tat and V2 loop-deleted Env (EnvV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID50 (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIVSF162P4cy ). Upon challenge, three of four macaques immunized with Tat and EnvV2, and two of three monkeys immunized with EnvV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvV2 or Tat/EnvV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvV2 alone, the Tat and EnvV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvV2 approached the immune response assessed after two inocula with the Tat/EnvV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvV2 or Tat/EnvV2 vaccination groups in this pilot study, vaccination with Tat/EnvV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvV2 as compared to EnvV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvV2 vaccine may afford superior protection against infection. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Currently, HIV/AIDS vaccine candidates based on the combination of structural and regulatory HIV proteins may hold the best

∗ Corresponding author. Tel.: +39 06 49903209; fax: +39 06 49903002. E-mail address: [email protected] (B. Ensoli). 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.02.006

promise for developing a protective vaccine [1]. In fact, the overall disappointing results of HIV/AIDS vaccine phase IIb/III clinical trials conducted to date [2,3] can be, at least in part, ascribed to the inadequacy of classical vaccine strategies based on HIV structural antigens (Env, and/or Gag, and Pol), which failed to provide protection [4]. On the other hand, less traditional vaccine approaches with HIV regulatory proteins (Tat, Rev, and Nef) were able to contain virus replication and preventing disease onset and progression

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in preclinical studies [1,4]. More recently, novel vaccine modalities encompassed both structural and regulatory HIV proteins to generate antibody (Ab) and cell-mediated immunity against multiple HIV components that are key for HIV entry/replication. Such combined vaccines are intended to block/control early and late HIV infection-related events, to contain acute virus infection and/or protect the host from disease progression [1,4,5]. The exploitation of HIV regulatory proteins’ immunomodulatory properties [6,7] represents an added value of this combined vaccine approach [1]. We describe here the rational design and evaluation in macaques of an innovative HIV/AIDS vaccine based on the combination of HIV Tat and Env proteins, aimed at inducing protective immunity capable of neutralizing the virus as well as Tat biological activities [8–11]. In particular, this study was conceived as a pilot study to evaluate the effects of native Tat protein addition to an Env-based vaccine. To this aim, the two pivotal experimental groups consisted of macaques immunized with Env only or with Tat and Env. Control groups included animals inoculated with the adjuvants only or left untreated (naïve), and one animal vaccinated with the native Tat protein alone. An oligomeric, SF162 strain-derived, V2 loop-deleted Env (EnvV2) was chosen for its capacity of eliciting neutralizing antibodies (nAbs) against primary HIV isolates [12]. The deletion of the V2 loop allows exposure of conserved neutralization-sensitive epitopes to increase the breadth of vaccine-elicited Ab responses, thus potentially circumventing the issue of HIV Env intra- and interclade variability [13]. The inclusion of biologically active, HIIIB strain-derived Tat protein in the vaccine regimen was intended to generate immune responses against the early viral product Tat and to exploit Tat immunomodulatory properties. Tat is key for virus replication, cell-to-cell virus transmission, and HIV pathogenicity, and can enter both infected and uninfected cells, modulating the expression of cellular genes [14]. Biologically active Tat promotes monocyte-derived dendritic cells maturation towards a Th-1 polarizing phenotype as well as their antigenpresenting activity, leading to a more efficient presentation of both allogeneic and exogenous soluble antigens, in vitro [15,16]. By modulating the composition and activity of the immunoproteasome, Tat changes the hierarchy of CTL epitopes in favor of subdominant and cryptic ones and shows vaccine-adjuvant properties [17–20]. Tat is highly conserved in its immunodominant domains representing, in principle, an ideal target for a broadly effective vaccine [21]. Because of its multiple functions, immune responses against biologically active Tat can contribute to the control of HIV infection and/or disease progression. Of note, anti-Tat cellular or humoral immunity was reported to correlate, in the course of natural infection, with early virus control [22,23] or with asymptomatic infection and long-term non-progression to AIDS [24,25], respectively. Furthermore, a retrospective analysis conducted on 112 cynomolgus macaques indicated that vaccination with the biologically active Tat protein reduced the rate of infection acquisition against challenge with 10 MID50 of pathogenic SHIV89.6P, contained acute CD4+ T cell depletion against 15 MID50 of virus, and contained CD4+ T cell loss in the chronic phase of infection, regardless of the challenge dose [26]. Recently, the HIV Tat vaccine has shown to be safe and immunogenic in preventive and therapeutic phase I trials [27,28] and to intensify the therapeutic effects of HAART in successfully treated HIV-infected individuals, making Tat a promising target of an HIV/AIDS vaccine [29]. The experimental design of this pilot study consisted of multiple subcutaneous and intranasal immunizations with proteins in adjuvants, followed by assessment of immune responses at both the systemic and the mucosal levels. The Alum adjuvant (alu-

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minum phosphate), which is commonly used in licensed human vaccines [30], was employed for subcutaneous immunizations, whereas adjuvant LT-K63 was given intranasally [31]. LT-K63 is an Escherichia coli heat-labile enterotoxin mutant, which proved generally safe and effective as an intranasal adjuvant both in animals and in humans [31–33]. In particular, multiple LT-K63 intranasal administrations in animals did not induce histological inflammatory changes in the respiratory tract or olfactory bulbs and in the meninges [31]. When co-administered with a large number of immunogens by different routes in mice, LT-K63 was able to enhance immune responses [31]. In female rhesus macaques immunized with the HIV Env protein and LT-K63 adjuvant either intramuscularly, or intramuscularly and intranasally, protection was achieved against intravaginal simian-human immunodeficiency virus (SHIV) challenge [33]. LT-K63 also showed an overall good safety profile and intranasal adjuvanticity for influenza in humans [32]. Nonetheless, more recently (after the completion of our monkey study), two phase I clinical trials [34] on nasal subunit vaccines against HIV and tuberculosis employing LT-K63 as an adjuvant confirmed previous concerns on the association of LT-K63-containing nasal influenza vaccine and Bell’s palsy [35]. Efficacy of the Tat/EnvV2 or EnvV2 alone vaccines was evaluated against homologous intrarectal challenge with an R5 SHIV.

2. Materials and methods 2.1. Macaques housing, immunizations, and virus challenge The male Mauritian cynomolgus monkeys (Macaca fascicularis) employed in this study, negative for simian immunodeficiency virus (SIV), STLV-1, simian type-D retroviruses, and simian Herpes B virus infections, were housed at the National AIDS Center, Istituto Superiore di Sanità (ISS), according to the European guidelines for non-human primate care (ECC, Directive No. 86-609, Nov. 24, 1986). Animal experiments were approved by the Quality and Safety Committee for Animal Trials of the ISS. All clinical procedures were performed upon anesthesia with 7–10 mg/kg Zoletil (a combination of Tiletamine and Zolazepem). All monkeys were observed daily for behavior, major local reactions after immunizations, and clinical signs of disease. Serum biochemical/hematological parameters and animal weight were assessed on a regular basis, according to the blood drawing schedule. No major local reactions were recorded after immunizations and all safety-related parameters evaluated were in the normal range at all times. All animals lived throughout the study. The Tat protein used for immunization (HIV-1 clade B, strain HIIIB, ABL) was expressed in E. coli and purified (>95% pure) as described previously [6,15]. Tat biological activity was verified by rescue assay using the HLM-1 cell line carrying a Tat-defective HIV provirus, as already published [6]. The V2-deleted oligomeric gp140 protein (EnvV2, derived from the HIV-1 clade B, strain SF162, Novartis) was produced in CHO cells and purified (approximately 95% pure) as previously reported [36]. The purified glycosylated oligomeric EnvV2 protein was mostly homogeneous and likely in trimeric conformation [36]. The native structure and functionality of the EnvV2 protein was confirmed by quantitative binding of EnvV2 to soluble CD4 and its structural integrity was verified by capture ELISA with a panel of monoclonal Env-specific antibodies as reported [36]. Stability studies assessed that the oligomeric EnvV2 protein is stable between 25 and 45 ◦ C [36]. Based on former immunization protocols with the Tat protein [26,37], and to vaccinate with roughly equimolar amounts, 10 ␮g of Tat protein and 100 ␮g of EnvV2 protein were used in this

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Fig. 1. (a–d) Experimental design. The study involved the following macaques: 4 vaccinees administered Tat and EnvV2 proteins (a), 3 monkeys immunized with EnvV2 (b), 1 macaque vaccinated with Tat alone (c) and 1 animal injected with the adjuvant only (d). At week 32, 2 naïve macaques were included in the study as additional controls. At week 96, all monkeys were exposed intrarectally to 35 MID50 of the SHIVSF162P4cy .

study. Four macaques belonging to the Tat/EnvV2 experimental group received two subcutaneous immunizations with the native Tat protein in Alum [30], at weeks 0 and 4, three subcutaneous immunizations with the combination of Tat and EnvV2 proteins in Alum, at weeks 12, 23 and 43, two nasal boosts with Tat and EnvV2 proteins in LT-K63 [31], at weeks 57 and 67, and one final subcutaneous boost with Tat and EnvV2 proteins in Alum, at week 87 (Fig. 1a). The initial two priming immunizations with

Tat only were aimed at avoiding the possible immunodominance of Env over the generation of immune responses to Tat [38–40]. The second experimental group consisted of three macaques that were administered the EnvV2 protein alone (Fig. 1b). Controls included (i) one animal vaccinated with the native Tat protein only (8 immunizations) (Fig. 1c) (ii) one macaque that received only the adjuvants (Alum or LT-K63) (Fig. 1d), and (iii) two naïve monkeys, which were enrolled 32 weeks after

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the beginning of the study and left untreated until the time of challenge. At week 96 after the first immunization, all vaccinated and control monkeys were exposed intrarectally to 35 MID50 (monkey infectious dose 50) of SHIVSF162P4cy . This challenge virus was obtained by passaging the R5 SHIVSF162P4 [41,42] in one Mauritianorigin cynomolgus macaque (AH595, intravenous infection) and by isolating the resulting virus on PHA-activated, CD8+ T-cell depleted macaque peripheral blood mononuclear cells (PBMC). The virus stock was then titrated in vivo by intrarectal exposure of 10 Mauritian cynomolgus macaques (titer: 1.8 × 102 MID50 /ml). 2.2. Detection of antibodies against Tat or Env Upon collection, saliva samples were added a cocktail of protease inhibitors (Boehringer Mannheim-Roche) and antibiotics. Rectal secretions were obtained by processing rectal swabs with an extraction buffer containing protease inhibitors and antibiotics. Plasma, saliva and rectal secretion samples were stored at −80 ◦ C. ELISA plates were coated with Tat or oligomeric EnvV2 proteins and the assay performed as reported previously [21] for the assessment of specific IgA, IgG, or IgM in plasma, saliva (IgG or IgA), and rectal secretions (IgG or IgA). Horseradish peroxidase (HRP)-conjugated goat polyclonal Ab to monkey IgA, reactive with both serum and secretory IgA (Accurate Chemical and Scientific Corporation), HRP-conjugated rabbit polyclonal Ab to monkey IgM (Accurate Chemical and Scientific Corporation), and HRPconjugated rabbit polyclonal Ab to monkey IgG (Sigma–Aldrich) were used to quantify specific immunoglobulins in duplicate serial dilutions of plasma, saliva or rectal secretions. In general, there was no evidence that specific Abs detected in mucosal samples were secreted locally, rather than transudated from blood. Known positive and negative monkey samples were used as internal assay controls in each plate. Non-antigen specific plasma reactivity was taken into account by also testing samples on “nonantigen coated” wells, where the Tat or EnvV2 proteins were replaced by their resuspension buffers. “Delta” optical density (OD) reading values to control for non-specific sample binding were obtained by subtracting OD readings of “non-antigen coated” wells from OD readings of antigen-coated wells for each sample dilution tested. Binding Ab (bAb) titers were assessed as the reciprocal highest sample dilution that gave OD readings >3 standard deviations above negative controls, consisting of a panel of pre-immunization samples. Cut-offs were calculated for both antigen-coated wells and the corresponding “delta” values. Samples were considered positive when both cut-offs were exceeded. Total IgA were also measured in saliva samples and rectal secretions with a commercial kit for the measurement of human IgA (Cygnus Technologies). Epitope mapping of bAbs (IgG) was similarly performed, upon coating plates with 250 ng/well of Tat peptides or 10 ng/well of biotinylated Env peptide pools (for Env epitope mapping, streptavidin-coated microplates were used) [21]. Selected plasma samples were also tested by Western blot assay (Alfa Wassermann HIV Blot 2.2). 2.3. Neutralization assay Plasma nAbs were assessed using a viral infectivity assay based on TZM-bl cells infected with replication competent or pseudotyped viruses [43–49]. Percent neutralization was calculated relative to the corresponding negative control infection, containing pre-immune plasma of the same monkey; nAb titers were esti-

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mated as the reciprocal plasma dilution giving 50% or 80% inhibition of infection (ID50 or ID80). To facilitate the understanding of potential differences among the animals, week 93 pre-challenge plasma neutralizing titers and percent neutralization curves against replication-competent HIVSF162 were also calculated for pre- and post-immunization samples relative to to the virus infection control of the assay for each monkey immunized with Tat/EnvV2 or EnvV2. Replication competent viruses were propagated in human PBMC, whereas pseudoviruses were produced by co-transfection of 293T cells with pSG3env and Env-containing plasmids. All virus stocks were made cell free by low speed centrifugation and filtration (0.45 ␮m) and were titrated in TZM-bl cells prior to performing neutralization assays. 2.4. ELISpot assays Anti-Tat or anti-Env cells producing IL-2 or IL-4 were enumerated by using Mabtech ELISpot kits. Briefly, Ficoll Hypaque-purified PBMC were seeded at 2 × 105 cells/well in duplicate wells of 96-well microtiter plates coated with monoclonal Abs (mAbs) specific for monkey IL-2 or IL-4. Cell cultures were incubated overnight with medium, 2 ␮g/ml PHA, and Tat or Env-derived peptides at a final concentration of 2 ␮g/ml for each peptide. Peptide pools composition: one pool for Tat (aa 1–86; 15mers overlapping by 11 aa); three (pool 1 = aa 1–306, pool 2 = aa 307–594, pool 3 = aa 591–891) or four (pool 1 = aa 1–262, pool 2 = aa 167–428, pool 3 = aa 417–676, pool 4 = aa 583–847) pools for Env (15mers overlapping by 11 aa). PBMC were then discarded and biotinylated anti-IL-2 or IL-4 mAbs were added to the wells followed by the addition of streptavidin-alkaline phosphatase and, finally, of a chromogenic substrate. After development, spots were counted using an ELISpot reader (A.EL.VIS). The background was calculated as twice the mean value of IL2 or IL-4 spot-forming cells (SFC)/1 × 106 cells in non stimulated samples. Samples yielding IL-2 or IL-4 SFC/1 × 106 cells greater than 50 after the subtraction of the background were scored as positive. 2.5. Proliferation assay Lympho-proliferative responses to Tat or Env were assessed as reported [37,50]. One peptide pool was used for Tat, whereas 3 pools were utilized for Env (pool 1 = aa 1–306, pool 2 = aa 307–594, pool 3 = aa 591–891). Stimulation indices (SI) ≥ 3 were considered positive [37,50]. 2.6. Assessment of plasma viral RNA and proviral DNA Plasma viral RNA and cell-associated proviral loads were measured as already described [51,52]. 2.7. Statistical analysis Given the low sample sizes, the assumption of a normal distribution of the data was not possible. The non-parametric Mann–Whitney test or the Wilcoxon matched-pairs signedranks test was therefore used in comparative analyses, as appropriate. In order to maximize the statistical power, when previous data were available to support the assumption that differences between groups were in one specific direction, one-tailed analyses were performed. In particular, our data (unpublished) obtained in mice immunized with Tat, EnvV2, or Tat/EnvV2 indicated that the Tat/EnvV2 vaccine may induce broader anti-Env bAb epitope specificity. On the other hand, previous results obtained

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Fig. 2. (a and b) Anti-Tat plasma IgM (a) and IgG (b) titers. Black arrows indicate the time of challenge; week numbering to the left of arrows is referred to weeks after the first immunization, whereas week numbering to the right of arrows is to be intended post-challenge. The dashed lines represent the lowest dilution tested (1/25 for IgM, 1/100 for IgG). All negative titers (<25 for IgM, <100 for IgG) are shown below the dashed line, lying on the x-axis. Pre- and post-challenge anti-Tat IgM and IgG responses of animals belonging to the EnvV2 or adjuvant/naïve groups remained negative, and are not shown.

in EnvV2-vaccinated macaques [33] also showed that higher nAb titers correlate with protection against mucosal SHIVSF162P4 challenge. As for the analysis of anti-V3 loop IgG reactivity levels in protected vs. infected monkeys, the choice of performing a one-tailed statistical analysis was inherent to the preliminary observation that all protected animals had OD readings >0.5, whereas infected macaques had OD readings <0.5. P values ≤0.05 were considered significant. It is worth noticing that when the total sample size is seven or less (as in several of the analyses performed in this study), the two-tailed Mann–Whitney test will always result in a P value >0.05, no matter how much the groups differ.

3. Results 3.1. Anti-Tat binding antibodies and epitope mapping All Tat-containing vaccine regimens elicited bAb responses against Tat, with peak titers ranging between 12,800 and 25,600. Two immunizations with Tat were sufficient to generate measurable anti-Tat IgM (Fig. 2a) and IgG (Fig. 2b) in all vaccinees. Before challenge, low-titer (titer: 25), Tat-specific IgA were found in the plasma of 3 (animals AC252, AC017, and AC259) of the 5 monkeys

immunized with Tat or Tat and EnvV2 combined, respectively (data not shown). Pre-challenge mucosal anti-Tat IgG remained undetectable for all animals until week 93 pre-challenge, when Tat-specific IgG were found (titer: 40) only in the saliva of macaque AC017, immunized with Tat/EnvV2, after the final subcutaneous immunization that was given after 2 intranasal vaccinations (data not shown). Pre-challenge mucosal Tat-specific IgA were undetectable in the saliva and rectal secretions of all vaccinees (data not shown). After 4 immunizations with Tat, plasma IgG specificity was directed mainly against the immunodominant Tat N-terminus (aa 1–15) and, to a much less extent, to epitopes in the core (aa 37–48), basic (aa 49–56) or glutamine-rich region (aa 57–73), as well as in the region (aa 74–86) containing the RGD domain (aa 78–80) responsible for binding to integrins (Fig. 3a). Of note, the fifth subcutaneous immunization strengthened and broadened the overall anti-Tat bAb specificity especially to the basic region, the glutamine-rich region and Tat RGD domain (statistical analysis performed by comparing week 26 vs. week 47 ELISA OD readings relative to all Tat peptides or on the number of Tat epitopes recognized by plasma IgG, for plasma of Tat- or Tat/EnvV2-vaccinees, respectively; two-tailed Wilcoxon matched-pairs signed-ranks test, P < 0.0001 and P = 0.063, respectively, Fig. 3a and b).

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Fig. 3. (a and b) Plasma anti-Tat IgG epitope mapping assessed in all macaques vaccinated with Tat, or Tat + EnvV2, at week 26 (a) or week 47 (b) pre-challenge. Plasma samples were tested on individual Tat peptides by ELISA. Blocks below the histograms represent Tat protein domains, as specified by flanking labels. PRR: proline-rich region; CRR: cysteine-rich region; CR: core region; BR: basic region; GRR: glutamine-rich region; RGD-IBR; RGD-integrin binding region. Optical density (OD) values exceeding the assay cut-off are shown. Plasma anti-Tat IgG titers are also displayed to the right of each epitope mapping bar graph. (c and d) Plasma anti-Env IgG epitope mapping assessed in all macaques vaccinated with EnvV2, or Tat + EnvV2, at week 26 (c) or week 93 (d) pre-challenge. Plasma samples were tested on 22 peptide pools containing 6 peptides each by ELISA. Numbered blocks below the histograms represent Env protein domains, as specified by flanking labels. C1: first conserved region; V1-V2: first and second variable loops; C2: second conserved region; V3-C3: third variable loop and third conserved region; V4-C4: fourth variable loop and fourth conserved region; V5-C5: fifth variable loop and fifth conserved region; Gp41-FP: Gp41 fusion peptide; Gp41-N-HR: Gp41 N-terminal heptad repeat region; Gp41-ID: Gp41 immunodominant region; Gp41-C-HR: Gp41 C-terminal heptad repeat region. OD values exceeding the assay cut-off are displayed. Plasma anti-Env IgG titers are also displayed to the right of each epitope mapping bar graph.

3.2. Anti-Env binding antibodies and epitope mapping Anti-EnvV2 bAbs were elicited in all vaccinees immunized with EnvV2. Interestingly, while monkeys vaccinated with EnvV2 developed measurable anti-Env IgM after two immunizations, a single injection with the combination of Tat and EnvV2 was sufficient to induce a detectable IgM response against Env (Fig. 4a). In contrast, one immunization with EnvV2 was sufficient to induce EnvV2-binding IgG in all vaccinees (Fig. 4b). Env-specific plasma IgA were detected in all vaccinees after 2 or 3 immunizations with EnvV2, with pre-challenge peak titers ranging from 50 to 400 (data not shown). Pre-challenge mucosal anti-Env IgG, which were undetectable at weeks 57, 59, 67, and 70, were found in saliva or rectal secretions of 3 out of 4 Tat/EnvV2 vaccinees (monkeys AC252, AF523, and AC017; titer range: 10–80), and in 2 of 3 macaques immunized with EnvV2 (monkeys AF837 and AF847; titer: 10), at week 93, 6 weeks after the last systemic immunization that followed 2

intranasal injections, and 3 weeks before challenge. Pre-challenge anti-Env mucosal IgA remained undetectable (data not shown). Env-specific plasma IgG epitope mapping performed at weeks 26 (Fig. 3c) and 45 (not shown) revealed that macaques immunized with the combined vaccine displayed a trend towards a broader specificity, namely higher numbers of Env regions recognized by plasma IgG (one-tailed Mann–Whitney test, P = 0.076), than monkeys vaccinated with EnvV2 alone. Although this difference in antibody response breath was no longer apparent at week 93, overall differences in epitope specificity remained appreciable between the two groups throughout the immunization period. In particular, at week 93 plasma IgG of animals in the EnvV2 group displayed a trend towards stronger specificity for the V1 loop (peptide pool 4) and V5 loop-C5 (peptide pool 16) domains of Env, as confirmed by comparative statistical analysis performed on plasma IgG epitope mapping data (OD readings) for monkeys belonging to the two groups (two-tailed Mann–Whitney test, P = 0.057 and P = 0.057, respectively) (Fig. 3d). In addition, as compared to monkeys immu-

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Fig. 4. (a and b) Anti-EnvV2 plasma IgM (a) and IgG (b) titers. Black arrows indicate the time of challenge; week numbering to the left of arrows is referred to weeks after the first immunization, whereas week numbering to the right of arrows is to be intended post-challenge. The dashed lines represent the lowest dilution tested (1/25 for IgM, 1/100 for IgG). All negative titers (<25 for IgM, <100 for IgG) are shown below the dashed line, lying on the x-axis. Macaques AG172 and AG337 were included in the study at the time of challenge; therefore, for these animals, antigen-specific plasma IgM and IgG titers were measured from that time point onwards.

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nized with EnvV2, macaques belonging to the Tat/EnvV2 group displayed an overall higher reactivity for epitopes within the V3 loop (peptide pool 10) region of Env (analysis performed by comparing anti-Env peptide pool 10 OD readings for plasma IgG of EnvV2- vs. Tat/EnvV2-vaccinees, at weeks 26, 45 and 93; twotailed Mann–Whitney test, P = 0.03; Fig. 3c and d). 3.3. Neutralizing antibody responses against homologous vaccine HIV or SHIV and cross-clade neutralization of heterologous primary HIV isolates All animals in the Tat/EnvV2 and EnvV2 groups mounted pre-challenge neutralizing Ab (nAb) responses against homologous vaccine HIVSF162 [53] (Fig. 5a and b, Supplemental Figure 1a) or to the derived pseudotyped virus (pHIVSF162 , data not shown) [54–56]. Macaques of both groups neutralized the challenge virus SHIVSF162P4cy (data not shown). When neutralization analysis was performed on pre-immune plasma relative to the virus infection control (see Section 2 for details), typical range variability among animals, in terms of non-specific interference with virus infection was observed (Supplemental Figure 1b). In line with this, the neutralization curve slopes obtained for week 93 pre-challenge immune samples relative to the virus infection control were shaped similarly to those calculated vs. the corresponding pre-immune plasma. This confirmed that differences among vaccine-induced nAb titers assessed in plasma of different animals were not substantially affected by pre-existing factors in plasma obtained before immunization (Supplemental Figure 1). Of note, early in the vaccination schedule, i.e. 3 weeks after the second immunization including EnvV2, a trend (two-tailed Mann–Whitney test, P = 0.057) was observed in favor of a higher ratio of HIVSF162 -nAb titers to anti-SF162 Env bAb titers in the Tat/EnvV2 vaccination group as compared to the EnvV2 group (Fig. 5a and b). When tested against heterologous primary HIV isolates of nonB clades, plasma from animals of both Tat/EnvV2 and EnvV2 groups showed some cross-clade neutralization (Table 1). 3.4. Cellular immune responses All monkeys immunized with Tat or Tat/EnvV2 proteins developed measurable pre-challenge anti-Tat specific cellular responses, as assessed by both lympho-proliferative assay (Fig. 6a) and ELISpot assays for IL-4 (Fig. 6b) and for IL-2 (Fig. 6c) production. On the other hand, pre-challenge Env-specific cellular responses were detected in several, yet not all, animals belonging to the EnvV2 or Tat/EnvV2 immunization groups (Fig. 6d–f). Overall, many cellular responses were undetectable at the time of challenge. 3.5. Outcome of challenge Upon challenge, all controls (1 adjuvant-inoculated animal, 2 naïve macaques, and monkey AC259, vaccinated with Tat) became systemically infected (Figs. 4, 5c, 6d, and 7c, d, k, l, Table 2 and Supplemental Figure 2). Despite both plasma and cellular viremia of naïve monkey AG337 remained undetectable, this animal showed unambiguous signs of systemic infection. In fact, AG337 mounted nAbs against HIVSF162 at week 24 post-challenge (Fig. 5c) and bAbs against HIVSF162 oligomeric EnvV2 (IgA, titer: 25, and IgG, titer: 200; Fig. 4b), and against HIV Gag (p24) and Env (gp120 and gp160) proteins (Table 2 and Supplemental Figure 2), as assessed at week 42 post-challenge by ELISA and Western blot assay, respectively. In contrast, 3 out of 4 monkeys immunized with Tat and EnvV2 combined, and 2 of 3 monkeys immunized with EnvV2 alone did not show any signs of infection (Fig. 7a and b), as also verified by

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viral DNA assessment in rectal and lymph node biopsy samples performed 14 weeks post-challenge (Fig. 7i and j). These data were also confirmed in Western blot assay, which revealed the presence of virus-induced anti-HIV Gag (p24) Abs in the infected vaccinees AC017 (Tat/EnvV2) and AF837 (EnvV2), and in viremic control animals AF318 (adjuvants) and AG172 (naïve), but not in protected vaccinees AF874, AC252, AF523, AF707, and AF847 (Table 2 and Supplemental Figure 2). The 5 macaques vaccinated with EnvV2 or Tat/EnvV2 that resulted protected from systemic infection displayed higher peaks of pre-challenge nAb titers (ID80) against HIVSF162 than the 2 infected vaccinees belonging to the same groups (pre-challenge peak nAb ID80 values occurred at week 26 for animal AC017, at week 47 for monkeys AF874, AC252, AF523, AF837, AF707, and at week 93 for animal AF847; one-tailed Mann–Whitney test, P = 0.048). Interestingly, such a correlation with protection was not observed when considering nAb titers assessed in vitro against the corresponding pseudotyped virus, which includes an HIVSF162 -derived Env protein. Moreover, 3 weeks before challenge (week 93), all protected monkeys exhibited stronger (OD >0.5) reactivity to peptides encompassing the Env V3 loop (peptide pool 10) than infected vaccinees AF837 and AC017 [comparison of ELISA OD readings of 5 protected vaccinees (all OD readings >0.5) vs. infected vaccinees (all OD readings <0.5), one-tailed Mann–Whitney test, P = 0.048, Fig. 3d]. In contrast, no clear correlation was found between the frequency or intensity of pre-challenge cellular responses and the outcome of virus challenge. All animals maintained normal levels of CD4+ cell counts (both percentage and absolute cell number) and lived throughout the 51-week follow-up (Fig. 7e–h) without showing obvious signs of disease.

4. Discussion In this study we showed that a vaccine based on the combination of HIV EnvV2 and native Tat proteins generated more rapid IgM responses and broader IgG epitope specificity than EnvV2 alone. Protection from mucosal infection was achieved in monkeys vaccinated with the Tat/EnvV2 combined vaccine or with EnvV2. These findings extend upon those reported using the EnvV2 protein alone, where vaccine protection was also observed in a somewhat different model in which female rhesus macaques were challenged intravaginally with the SHIVSF162P4 [33]. The immunization schedule followed here was designed to circumvent the immunodominance of the Env protein on Tat, observed in the course of natural infection [24,25,27,57–62], which may potentially impact the immune response to Tat in a Tat/EnvV2 vaccine approach [38,40]. For this reason, Tat was administered subcutaneously in Alum twice before the animals received the combined vaccine. In order to improve mucosal immunity, the vaccine was also given intranasally twice. The effect of two intranasal immunizations on mucosal immunity was not immediately appreciable. However, even though pre-challenge antigen-specific mucosal IgA were not found in saliva and rectal secretions, at any time points, anti-Tat and anti-Env IgG became detectable in mucosal samples of several animals after the final subcutaneous immunization that followed mucosal vaccine administration. Although this may merely be the result of repeated systemic immunizations (six in total), an effect by LTK63-adjuvanted nasal vaccinations on mucosal immunity cannot be ruled out. Noteworthy, repeated immunizations with Tat or Tat/EnvV2 strengthened and broadened Ab responses against the Tat basic region, the glutamine-rich region and the RGD-containing domain, rather than to the N-terminus of Tat, which is its main immun-

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a

F. Ferrantelli et al. / Vaccine 29 (2011) 2918–2932

b

Tat + ΔV2Env

ΔV2Env

c

Control

Fig. 5. (a–c) Plasma neutralizing antibody titers measured against HIVSF162 (ID50 and ID80 – bars) and anti-EnvV2 binding antibody titers (line) for each monkey of the Tat + EnvV2 (a), EnvV2 (b), or control (c) groups.

odominant region [63,64]. This suggests that multiple vaccinations with Tat may be useful for eliciting an optimal immune response against biologically active Tat and its domains that exert pleiotropic effects [8–11,14–16,65]. Anti-Env bAb responses in monkeys vaccinated with EnvV2 or Tat/EnvV2 differed in both the kinetics of Ab appearance and in the specificity. In fact, Env-directed IgM became

detectable earlier in the combined vaccine group than in EnvV2immunized animals. In addition, after 3 immunizations with EnvV2 or Tat/EnvV2, anti-Env IgG specificity was broader in the Tat/EnvV2 group. Differences in the anti-Env epitope specificity between groups persisted even after the sixth and last immunization, when monkeys vaccinated with Tat/EnvV2 displayed a strong specificity for the V3 loop-C3 region of Env, whereas plasma

Table 1 Pre-challenge neutralization of non-clade B HIV primary isolates. Immunization group

Tat + EnvV2 Tat + EnvV2 Tat + EnvV2 Tat + EnvV2 EnvV2 EnvV2 EnvV2 Tat Adjuvants Naive Naive

Monkey

AF874 AC252 AF523 AC017 AF837 AF707 AF847 AC259 AF318 AG172 AG337

ID50 (% neutralization at 1/30 plasma dilution) HIV-1 92UG037 clade A (R5)

HIV-1 97ZA009 clade C (R5)

HIV-1 92UG005 clade D (R5)

Week 26

Week 45

Week 26

Week 45

Week 26

Week 45

<30 (49%) <30 (30%) 250 (69%) <30 (39%) <30 (−14%) <30 (−48%) <30 (8%) <30 (46%) <30 (8%) ND ND

<30 (45%) 36(52%) <30 (44%) 60(72%) <30 (48%) <30 (36%) 64 (64%) <30 (35%) <30 (35%) ND ND

<30 (6%) <30 (14%) 74 (70%) <30 (14%) <30 (−31%) <30 (−10%) <30 (21%) <30 (−8%) <30 (21%) ND ND

<30 (23%) <30 (49%) <30 (26%) 35 (56%) <30 (47%) 40 (58%) <30 (44%) <30 (6%) <30 (25%) ND ND

<30 (20%) <30 (21%) 46 (85%) <30 (−29%) <30 (0%) <30 (−39%) <30 (42%) <30 (38%) <30 (−5%) ND ND

44 (58%) <30 (−15%) <30 (16%) <30 (48%) <30 (18%) <30 (29%) 53 (82%) <30 (10%) ND ND ND

Positive results are highlighted in bold. ND: not done.

F. Ferrantelli et al. / Vaccine 29 (2011) 2918–2932

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Lympho-proliferave responses to Tat

a AF874 Tat + Env ∆V2 AC252 Tat + Env ∆V2 AF523 Tat + Env ∆V2 AC017 Tat + Env ∆V2

50

50

AC259 Tat 40

40

30

SI

SI

30 20

20

10

10 0

0

-6 4 8 12 17 23 27 32 38 45 70 87 0 2 8 14 24 42

0 4 8 12 17 23 27 32 38 45 70 87 93 0 2 4 8 14 24 42

Weeks

Weeks

An-Tat IL-4-producing cells

b AF874 Tat + Env ∆V2 AC252 Tat + Env ∆V2 AF523 Tat + Env ∆V2 AC017 Tat + Env ∆V2

AC259 Tat 600

IL-4 SFC/1 x 10^6 cells

IL-4 SFC/1 x 10^6 cells

600 500 400 300 200 100

500 400 300 200 100 0

0

12 17 23 26 32 45 47 70 87 93 0

8 12 17 23 26 32 45 67 70 87 93 0 2 4 8 24 42

8 24 42

350

350

IL-2 SFC/1 x 10^6 cells

300 250 200 150 100 50 0

AF837 Env ∆V2 AF707 Env ∆V2 AF847 Env ∆V2

300 250 200 150 100

300

70

87

93

0

2

4

8

24

200 150 100 50

50

42

AC259 Ta

250

0

67

0 67

70

87

93

0

Weeks

2

4

8

24

42

67

70

87

Weeks

AF874 Tat + Env ∆V2 AC252 Tat + Env ∆V2 AF523 Tat + Env ∆V2 AC017 Tat + Env ∆V2

50 40

AF837 Env ∆V2 AF707 Env ∆V2 AF847 Env ∆V2

(62) 50

93

0

2

4

8

24

42

Weeks

Lympho-proliferave responses to Env

d

50 AC259 Tat

40

40

30

SI

SI

SI

30

30

20

20

20

10

10

0

0

e

10

0 0 4 8 12 17 23 27 32 38 45 70 87 93 0 2 4 8 14 24 42 Weeks

0 4 8 12 17 23 27 32 38 45 70 87 93 0 2 4 8 14 24 42 Weeks

0 4 8 12 17 23 27 32 38 45 70 87 0 2 8 14 24 42 Weeks

An-EnvIL-4-producing cells

250

AF874 Tat + Env ∆V2 AC252 Tat + Env ∆V2 AF523 Tat + Env ∆V2 AC017 Tat + Env ∆V2

200 150

250

IL-4 SFC/1 x 10^6 cells

IL-4 SFC/1 x 10^6 cells

IL-2 SFC/1 x 10^6 cells

AF874 Tat + Env ∆V2 AC252 Tat + Env ∆V2 AF523 Tat + Env ∆V2 AC017 Tat + Env ∆V2

350

IL-2 SFC/1 x 10^6 cells

4

An-Tat IL-2-producing cells

c

100 50

AF837 Env ∆V2 AF707 Env ∆V2 AF847 Env ∆V2

200 150 100 50 0

0

8 12 17 23 26 32 45 67 70 87 93 0 2

8 12 17 23 26 32 45 67 70 87 93 0 2 4 8 24 42

4

8 24 42

Weeks

Weeks

An-EnvIL-2-producing cells

f

AF874 Tat + Env ∆V2

250

AF523 Tat + Env ∆V2 200

AC017 Tat + Env ∆V2

150 100 50

IL-2 SFC/1 x 10^6 cells

AC252 Tat + Env ∆V2

250

IL-2 SFC/1 x 10^6 cells

2

Weeks

Weeks

AF837 Env ∆V2 AF707 Env ∆V2 AF847 Env ∆V2

200 150 100 50 0

0 57

59

67

70

87

93

0

Weeks

2

4

8

24

42

57

59

67

70

87

93

0

2

4

8

24

42

Weeks

Fig. 6. (a–c) Anti-Tat or (d–f) anti-Env antigen-specific (a and d) lympho-proliferative responses, (b and e) IL-4-producing cells, or (c and f) IL-2-producing cells for macaques belonging to the Tat + EnvV2, EnvV2, or Tat groups. Black arrows indicate the time of challenge; week numbering to the left of arrows is referred to weeks after the first immunization, whereas week numbering to the right of arrows is to be intended post-challenge. Dashed lines indicate the cut-off value [50 spot-forming cells (SFC)/1 × 106 cells, exceeding the background]. Values below the dashed line are to be considered negative. The following pre- and post-challenge responses remained negative at all times and are not shown: anti-Tat lymphoproliferative responses and anti-Tat IL-4-producing cells of animals belonging to the EnvV2 or adjuvant/naïve groups, anti-Env lympho-proliferative responses of animals in the adjuvant/naïve group, anti-Env IL-4-producing cells of animals belonging to the Tat or adjuvant/naïve groups, anti-Tat or anti-Env IL-2-producing cells of animals belonging to the adjuvant/naïve group, and anti-Env IL-2-producing cells of the monkey vaccinated with Tat.

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AF523

b

AC017

5 4 3 2 Neg

1 0

AF707

c

AF847

5 4 3 2 Neg

1 0

0

10

20 30 40 Weeks post-challenge

50

5 4 3 2

10

20 30 40 Weeks post-challenge

50

2000

CD4+ T c cells/ul

2000

CD4+ T c cells/ul

2000

1000 500

7

17 27 37 Weeks post-challenge

47

-3

7

17

37

20 30 40 Weeks post-challenge

-3

7

17 27 37 Weeks post-challenge

1500

500 0 7

17 27 37 Weeks post-challenge

47

Proviral Copies (copies/μg DNA)

Neg

3

Tat

AC259

59

20

Neg

Neg

Neg

Neg

Env ΔV2

AF837

Env ΔV2

AF707

AF523

Neg

Neg

Env ΔV2

AF847

AC017

114

17

47

l Proviral Copies (copies/μg DNA)

Immunization Group

Neg

50

1000

Rectum

Neg

Tat + Env ΔV2

10

Lymph nodes

Neg

Tat + Env ΔV2

0

Monkey

Neg

AF874 AC252

Neg

1

Immunization Group

Lymph nodes

Tat + Env ΔV2

2

Rectum

Monkey

Tat + Env Δ V2 ΔV2

3

2000

500

k

Immunization Group

Lymph nodes

1000

Proviral Copies (copies/μg DNA)

Rectum

Monkey

4

50

1500

-3

47

j Proviral Copies (copies/μg DNA)

Immunization Group

27

5

2500

Weeks post-challenge

i

20 30 40 Weeks post-challenge

0

0 -3

AG337-Naïve

h 2500

0

10

g

1500

AG172-Naïve

6

0 0

2500

500

AF318-Adjuvants

0 0

f

1500

Neg

1

2500

1000

d

AC259 6

F. Ferrantelli et al. / Vaccine 29 (2011) 2918–2932

e CD4+ T c cells/ul

AF837 6

C t l Control

CD4+ T cells/ul

AC252

T Tatt

Log plasma viral RNA load (copies/ml)

Log plasma viral RNA load (copies/ml)

AF874 6

Log plasma viral RNA load (copies/ml)

a

ΔV2E ΔV2Env

Log plasma viral RNA load (copies/ml)

T t+E Tat EnvΔV2 ΔV2

Monkey

Lymph nodes

Rectum

Control - Adjuv.

AF318

42

12

Control - Naive

AG172

90

26

Control - Naive

AG337

Neg

1

Fig. 7. (a–d) Plasma viral RNA loads, (e–h) peripheral absolute CD4+ T-helper cell counts, and (i–l) proviral DNA copies in lymph node or rectum biopsy samples for animals belonging to the Tat + EnvV2, EnvV2, Tat, or control groups. (a–d) For plasma viral loads, the sensitivity of the assay (50 copies/ml) is indicated by the dashed line. Negative values are shown below the dashed line. (i–l) For proviral DNA in biopsy samples, the sensitivity of the assay was 1 copy per ␮g of DNA.

F. Ferrantelli et al. / Vaccine 29 (2011) 2918–2932

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Table 2 Western blot assay scoring. Immunization group

Monkey

Week post-challenge

EnvV2/Tat

AF874

8 42 8 42 8 42 8 42 8 42 8 42 8 42 8 42 8 42 8 42

AC252 AF523 AC017 EnvV2

AF837 AF707 AF847

Adjuvants

AF318

Naïve

AG172 AG337

HIV-1 proteins p17

p24

p31

p39

gp41

p51

p55

p66

gp120

gp160

− − − − − − + − − − − − − − ++ ++ ++ ++ − −

− − − − − − ++ ++++ ++ + − − − − +++ +++ ++++ +++ − +

− − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − −

+ + + + + + +++ +++ ++ + + − + + ++ ++ ++ ++ − −

− − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − −

− − − − − − − − − − − − − − − − − − − −

+ + ++ + + + ++++ ++++ ++++ ++ ++ ++ ++ + +++ +++ +++ ++++ − +

++ ++ ++ ++ ++ + ++++ ++++ ++++ +++ ++ ++ ++ + ++++ ++++ +++ ++++ − ++

samples from EnvV2-immunized animals showed a greater specificity for epitopes contained in the V1 loop and the V5 loop-C5 region. Differences between the EnvV2 or Tat/EnvV2 groups were also evident at early time points (after 2 immunizations with EnvV2 or Tat/EnvV2) in terms of anti-Env nAb/binding IgG ratios, with the Tat/EnvV2 combination vaccine group showing a greater relative presence of virus neutralizing responses. The above-described differences between responses elicited by the EnvV2- or Tat/EnvV2-based vaccines may be the result of the immunomodulatory and adjuvant properties of biologically active Tat protein, which might have modified Env processing by antigen presenting cells [15,16,19]. However, most differences between the EnvV2 and the Tat/EnvV2 vaccination groups became less appreciable as the number of immunizations increased, possibly because of the effect of immune responses previously elicited by vaccination. Abs directed against Tat may have modified the immunomodulation and adjuvanticity of the injected Tat protein [15,16,19], at the same time, both anti-Tat and anti-Env Abs may have modified the immunogenicity of subsequently administered vaccine components. By binding to their cognate proteins, the pre-existing vaccine-specific Abs may mask the main immunodominant determinants of Tat and EnvV2 thus preventing further boosting of responses against these regions, while allowing the broadening of Ab epitope specificity [66], as observed upon repeated immunizations in macaques administered with Tat or EnvV2 alone. The binding of specific Abs to the newly injected vaccine immunogen is also likely to have altered its structure [67] thus exposing otherwise subdominant and cryptic epitopes. This may explain why, after multiple immunizations, vaccinees administered with EnvV2 alone displayed a broadening of Ab specificity that resembled more those achieved earlier in the Tat/EnvV2 group. Overall, the co-administration of native Tat protein with EnvV2 might induce protective immunity in a more efficient/rapid fashion. Interestingly, EnvV2 and Tat/EnvV2 vaccinated monkeys that were protected from mucosal virus challenge displayed higher pre-challenge anti-HIVSF162 nAb peak titers (ID80) than infected macaques immunized with EnvV2 and Tat/EnvV2 in this study. The correlation between protection from infection and higher nAb titers assessed against the homologous replication competent virus rather than against the correspondent pseudotype may be due to

differences between the two viruses used, such as the genetic background (HIVSF162 or HIVPSG3Env ) and/or the virus stock preparation (propagation via infection of human PBMC vs. transfection of 293T human renal epithelial cell line), or to discrepancies between the neutralization assays with a replicating virus or with a pseudotyped single-cycle virus [45,68,69]. Our results showed that the nAb titers assessed by a virus neutralization assay based on the vaccinehomologous, replication-competent virus strain prepared in PBMC may be a better prognostic factor for the outcome of challenge than titers assessed by a pseudotyped virus-based assay. In this study, vaccinees that were protected against infection displayed higher pre-challenge levels of anti-Env V3 loop bAbs. Although no direct evidence is available that this bAb subset actually contributed to protection from SHIV challenge, an association between higher anti-Env V3 loop antibodies and protection is conceivable. In fact, the third variable region of Env, which is involved in HIV co-receptor binding and influences viral tropism [70–72], is highly immunogenic and able to induce broadly reactive, crossneutralizing Abs against its own conserved structures [73]. Of note, the V2-loop deletion present in the Env protein used in our study is known to enhance the natural immunogenicity of the V3 loop [13], which was even augmented here by the presence of biologically active Tat protein as a co-immunogen (see above). Lympho-proliferative, IL-2, and IL-4 cellular responses to Tat or Env were overall sporadic and low or undetectable at the time of challenge. Vaccine-specific cellular immunity and the outcome of virus challenge did not show any correlation. Of course, we cannot rule out the possibility that low assay sensitivity may have led to underestimation of actual cellular responses and, therefore, to the inability to find any cellular correlates of protection. We previously reported that a replication-competent adenovirus (Ad)-HIV recombinant prime/protein boost regimen based on HIV89.6P Env (gp140CFI) and HIVIIIB Tat reduced chronic viremia and preserved CD4+ T cells in rhesus macaques challenged intravenously with SHIV89.6P [5]. Despite in that study all animals became infected, higher anti-Tat and anti-Env bAb titers correlated with a better control of chronic viremia. Similarly, the present study corroborates the importance of Ab responses against infection, in particular the role of nAbs and anti-Env V3 loop bAbs in protection from mucosal virus challenge. Differences between the two studies, such as the absence of complete protection from infection in the Ad prime/protein boost-based study and the correlation with anti-Tat and anti-Env bAb rather than nAb titers, may be ascribed

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F. Ferrantelli et al. / Vaccine 29 (2011) 2918–2932

to the different immunogens, immunization regimens, viruses and routes of challenge, and nonhuman primate species utilized. Recent studies reported significant effects of MHC haplotypes of Mauritian cynomolgus macaques on susceptibility to SHIV infection [26,74,75], as well as SIV infection [76]. In particular, results from our recent and relatively large retrospective study conducted in 112 cynomolgus macaques of Mauritian origin indicate that the MHC background in addition to inherently influence the susceptibility to infection or progression to disease, significantly affects also the development of protective responses to vaccination, either positively or negatively [26]. However, all monkeys from that study were challenged intravenously with the X4-tropic SHIV89.6Pcy243 , and it is currently unknown whether those findings may be extended to macaques challenged mucosally with an R5-tropic SHIV, as reported here. In fact, differences among these studies highlighted the possible dependence of results on the characteristics of the challenge virus that is being considered. Therefore, at present, it is not possible to draw any substantiated conclusions on the potential relationship between MHC and susceptibility to SHIVSF162P4cy infection. We are currently conducting a monkey population study that includes a sufficiently large group of cynomolgus macaques infected with the R5 SHIVSF162P4cy to evaluate a potential role of MHC haplotypes in this model of infection. Compared to EnvV2 alone, the Tat/EnvV2 combined vaccine elicited Ab responses that were more rapid to develop and showed greater breadth after few immunizations. Ad hoc designed studies will be conducted to further corroborate and exploit the benefits of adding the biologically active Tat protein to the EnvV2-based vaccine. To verify whether the Tat/EnvV2 vaccine also induce an enhanced protection against infection, new studies will include larger vaccination groups and a smaller number of immunizations with the combined vaccine (or the relative single components) followed by mucosal challenge with homologous or heterologous SHIV. Importantly, biologically active Tat and EnvV2 proteins have already undergone phase I clinical testing as single vaccine components, proving safe and immunogenic [11,27,28,58,59] (http://www.hiv1tat-vaccines.info). In particular, the promising results obtained with both the therapeutic and the preventive trials with the Tat vaccine [11,27,28,58,59] provided the basis for advancing it to phase II trials both in Italy and in South Africa, respectively. An ad hoc exploratory interim analysis of the results from the ongoing therapeutic phase II trial with the Tat vaccine showed significant amelioration of several biochemical and cellular parameters accompanied by a progressive increment of CD4+ T cells, including regulatory T cells, and B cells with reduction of CD8+ T cells and NK cells in HIV-infected individuals [29]. The reduction of immune activation was accompanied by increases of CD4+ and CD8+ T cell responses against Env and recall antigens [29]. Taken together, these findings and the preliminary results reported here for the Tat/EnvV2 combined vaccine suggest that the Tat/EnvV2 vaccine may be a valuable candidate to be further explored in pre-clinical and clinical trials (http://www.hiv1tatvaccines.info).

Acknowledgements The authors thank Piergiorgio Pupino Carbonelli and the monkey facility staff of the National AIDS Center (Istituto Superiore di Sanità) for animal care and for all animal procedures, Claudia Rovetto for performing Western blot assays, Iole Macchia, Michela Sabbatucci, and Giulia Cencioni for assistance with the ELISpot assays, Pasqualina Leone, Gaia Sciaranghella, and Zuleika Michelini for help with the assessment of Ab responses, Domenico Fulgenzi, Daniela Compagnoni, Viviana Buffa, Martina Borghi, and Roberto

Marinelli for help with animal specimen processing, Emanuele Fanales-Belasio for help with cellular response data analysis, Silvia Baroncelli, Donatella Negri, and Barbara Ridolfi for assistance with data collection, Stefania Bellino for help with statistical analysis, Guendalina Fornari Luswergh for editorial assistance. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: SHIV SF162P3 env (15-mer) Peptides – Complete Set (used for the assessment of anti-Env cellular responses); TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc., pSG3env from Drs. John C. Kappes and Xiaoyun Wu, HIV-1SF162 from Dr. Jay Levy and pCAGGS SF162 gp160 from Drs. L. Stamatatos and C. ChengMayer, HIV-1 92UG037 and HIV-1 92UG005 from The UNAIDS Network for HIV Isolation and Characterization, HIV-1 97ZA009 from Dr. Robert Bollinger and the UNAIDS Network for HIV Isolation and Characterization (used for the evaluation of nAbs). This work was supported by: the AIDS Vaccine Integrated Project (AVIP), European Commission, grant LSHP-CT-2004503487; the Italian Concerted Action on HIV-AIDS Vaccine Development (ICAV), Italian National AIDS Program, Ministry of Health, Italy; the Joint Program ISS/Chiron for the development of a combined vaccine against HIV/AIDS. I.K.S. and S.W.B. were supported by HIVRAD grant 5 P01AI066287-03 from the NIAID-NIH. Conflict of interest statement: I.K.S. and S.W.B. are both employees of Novartis Vaccines and Diagnostics, Inc., and may be perceived to have a financial interest in the vaccines described herein.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.vaccine.2011.02.006.

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