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The Journal of Immunology, 2004, 172: 3745-3757.
Copyright © 2004 by The American Association of Immunologists

Control of Simian/Human Immunodeficiency Virus Viremia and Disease Progression after IL-2-Augmented DNA-Modified Vaccinia Virus Ankara Nasal Vaccination in Nonhuman Primates1

Frederic M. N. Bertley*,{dagger}, Pamela A. Kozlowski*,{dagger}, Shainn-Wei Wang*,{dagger}, Joseph Chappelle*, Jignesh Patel*, Oluwakemi Sonuyi*, Gail Mazzara{ddagger}, David Montefiori§, Angela Carville, Keith G. Mansfield and Anna Aldovini2,*

* Department of Medicine, Children’s Hospital, and {dagger} Department of Pediatrics, Harvard Medical School, Boston, MA 02115; {ddagger} Therion Biologics, Cambridge, MA 02142; § Department of Surgery, Duke University, Durham, NC 27710; and New England Regional Primate Research Center, Harvard Medical School, Southborough, MA 01772


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-{gamma}-secreting cells and TNF-{alpha}-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies of virus-specific immune responses in HIV-exposed and -infected individuals and of SIV vaccines in monkeys suggest that it is unlikely that vaccine approaches that stimulate a single arm of the immune system will provide effective protection from viral exposure (1, 2). It is possible that a combination of both mucosal (humoral and cellular) responses and systemic responses might result in protection from infection. Indeed, this combination has been suggested as key to resistance to HIV infection in highly exposed sex workers in Nairobi and Northern Thailand (3, 4). A similar role for combined HIV-specific mucosal IgA and systemic cellular responses in HIV protection has been proposed by Clerici et al. (5), studying discordant heterosexual couples.

Although it is known that Ag given via one route (mucosal or parenteral) might lead to responses of different magnitude in the mucosal and peripheral immune compartments, it is unclear whether high-level responses can be achieved in both. Data from mucosal immunization in human and nonhuman primates suggest that IgA responses to vaccines are maximal at the site of mucosal exposure, but distal responses are absent or weak. These observations support the notion of a compartmentalized mucosal immune system (6). Of all the mucosal routes tested in human and nonhuman primates, only vaccination via the nasal route has stimulated disseminated humoral and cellular mucosal responses (6, 7, 8). For instance, nasal immunization provides better vaginal responses than rectal immunization and can elicit rectal responses as well (8, 9). When compared with other mucosal routes, nasal vaccination has also been found to introduce greater systemic Ab responses. Thus, if immunity is desired at both mucosal and systemic sites, vaccination via the nasal route may be better than other mucosal routes (8).

Stimulation of SIV-specific mucosal responses has been achieved with different vaccine regimens that can induce both mucosal and systemic immunity, and in some instances protection from chronic systemic infection has been observed (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Induction of CTL able to home to mucosal sites is likely to be an important characteristic of a successful HIV vaccine. Live attenuated SIV strains, which preferentially replicate in activated T cells present in the extensive gut mucosal T cell pool, are particularly effective at inducing CTL that home to mucosal tissues (24).

Viral genomes that produce noninfectious virus-like particles have several features that make them attractive candidates for an AIDS vaccine. They may be capable of engendering immunity qualitatively similar to that obtained with attenuated viral vaccines, without establishing the persistent infection associated with attenuated viruses (23, 25). These noninfectious viral particles incorporate Env proteins and bind the viral receptor on target cells as well as the wild-type virus. Binding of the receptor is thought to trigger conformational changes in the Env protein that may reveal critical neutralizing epitopes on both gp120 and gp41. Exposure of these epitopes may not occur with Env-based vaccine preparations where the Env protein is not part of a virus particle.

Several SIV DNA vaccine constructs have been evaluated for their ability to induce protection against challenge with SIV (26, 27, 28, 29, 30, 31, 32, 33, 34). When challenged i.v., the animals sometimes resisted the establishment of chronic infection, and more frequently achieved a decreased viral load and had a more prolonged asymptomatic infection (26, 27, 28). A recent vaccine approach that investigated priming with DNA followed by recombinant adenovirus, both given i.m., has provided the most significant systemic cell-mediated responses observed to date following vaccination, as well as prolonged control of viremia after i.v. challenge (35). These studies represent important steps in the study of SIV DNA vaccines, but were not designed to evaluate mucosal immunity, and the challenge did not involve mucosal exposure. Only recently has SIV or simian/human immunodeficiency virus (SHIV)3 DNA been investigated as a mucosal immunogen (21, 23, 36).

Although the results obtained thus far with DNA vaccination in animal models are promising, there is clearly a need to increase the potency of this technology. A study conducted in nonhuman primates immunized i.m. demonstrates that the administration of a plasmid expressing human IL-2/Ig can substantially augment vaccine-elicited humoral and cellular immune responses (37). A similar study showed that IL-2 or IL-4 could increase the humoral responses to HIV Ags upon i.m. vaccination of primates (38). Given that mucosal responses, in addition to systemic responses, might be necessary for protection against sexually transmitted pathogens such as HIV, and that cytokines might be useful to enhance the immunostimulatory properties of a mucosal vaccine, it may be beneficial to identify a cytokine that enhances mucosal immune responses in primates.

We report here the results of a vaccine protocol that investigated a combined DNA-modified vaccinia virus Ankara (MVA) nasal immunization in rhesus macaques and show that this route of vaccination stimulates antiviral immunity at mucosal and parenteral sites. The levels of antiviral systemic immunity achieved by the vaccination were sufficient to prevent AIDS in all of the animals of one vaccine group.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal vaccines

The DNA plasmid pVacc4 used in the vaccination is a derivative of pVacc1 (23). pVacc1 includes a full SIVmac239 genome with multiple mutations in the NC basic domain and the functional domains of RT and INT, under the control of the CMV promoter. A 3.1-kb SphI-NcoI fragment that includes the env gene from pSHIV-KB9–3' (catalog no. 4129; National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, MD) replaced the corresponding SphI-SnaBI fragment of pVacc1 that includes the SIV env of SIVmac239. In addition, a stop codon replaced the initiation codon of the vpr gene. The DNA sequence was confirmed by sequencing, and the profile of viral particles produced by pVacc4 was evaluated by Western blot using macaque SIV-positive and human HIV-positive sera followed by 125I-labeled protein A. rMVA expressing SIV Gag-Pol and HIV Env proteins was prepared as previously described (39, 40). The IL-2/Ig plasmid was previously described (37). The rhesus macaque IL-12 expression plasmid was derived from the plasmid pSFG.hIL12.p40.L{Delta}p35 (41), which expresses human IL-12, by substituting the sequences encoding the human p40 and p35 subunits with the corresponding rhesus macaque sequences (42), positioned in the same configuration to produce plasmid pRM.IL-12.p40-p35. In this plasmid, the IL-12 p40 and -30 subunits are produced as a fusion protein in which the p35 subunit, deleted of its leader sequence, is fused to the p40 subunit by a Gly6Ser linker. IL-12 production by rmIL-12.p40.{Delta}Lp35 was tested in 293T transfection supernatant by ELISA (IL-12 p70 assay; R&D Systems, Minneapolis, MN), and its biological activity was measured according to the assay described in Ref. 41 .

Immunization and challenge

Male rhesus macaques were cared for at the New England Regional Primate Research Center using approved protocols under the guidelines established by the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Twenty animals were divided into four groups: pVacc4 DNA plus rMVA (group 1; n = 5); pVacc4 DNA plus IL-2/Ig DNA plus rMVA (group 2, n = 5); pVacc4 DNA plus IL-12 DNA plus rMVA (group 3, n = 5); and naive controls (group 4, n = 5). DNA was administered nasally as drops of a DNA solution in saline. For this administration, the vaccine DNA was formulated at a concentration of 10 mg/ml, and each DNA immunization consisted of 1.5 mg of DNA administered as a 150-µl dose, 75 µl in each nostril of the animal. Concentration and volumes were chosen to maximize the amount of DNA delivered in the smallest volume possible to facilitate retention of vaccine in the nasal cavity. The three DNA doses were administered on day 1 of wk 1, wk 9, and wk 25. A total of 109 PFU of rMVA, expressing SIV gag and pol and HIV env genes, were delivered to the nasal mucosa in volumes of 100 µl per nostril on wk 33. Two animals in each of these groups were Mamu-A*01 positive as determined by PCR. At wk 41, each animal was inoculated with a lethal dose (10 50% animal infectious doses; titrated via the rectal route) of pathogenic SHIV89.6P virus, administered nontraumatically with a needleless tuberculin syringe as cell-free virus in the rectum.

ELISA for anti-SIV and anti-HIV Env Abs

Plasma obtained by standard venipuncture before and at intervals after vaccination was analyzed by ELISA for antiviral IgG Abs. Both antiviral and total IgA in rectal secretions collected with Weck-Cel Surgical Spears (sponges; Medtronic Solan, Jacksonville, FL) were measured by ELISA. Blood contamination in secretions was assessed through measurement of hemoglobin with ChemStrips 4 (Boehringer Mannheim, Indianapolis, IN) as described (43). Hemoglobin in secretions was, on average, 0.06% of that in blood. The collection method and procedure for secretion extraction have been described (43). Total IgA was measured as described (23), with the exception that unlabeled and biotinylated purified polyclonal goat anti-monkey IgA Abs (both Rockland, Gilbertsville, PA) were used as the coating and secondary reagents, respectively.

ELISAs for antiviral Abs were performed as described (23). Briefly, plates were coated with 250 ng of SIVmac251 viral lysate (Advanced Biotechnologies, Columbia, MD), 100 ng of HIV-1 rgp120MN, or 100 ng of HIV-1 rgp41MN (both ImmunoDiagnostics, Woburn, MA). In ELISAs for Ag-specific IgA, 12 wells of each plate were coated with serial dilutions (6–200 ng/ml) of macaque IgA that had been purified in the laboratory from breast milk (Yerkes Primate Center, Atlanta, GA). After blocking, plates were loaded with plasma samples and positive control or with secretions and buffer (macaque IgA wells). Positive controls for antiviral IgG were purified human polyclonal anti-HIV p24 (cross-reactive with SIV p27), gp120, or gp41 IgG Abs (all ImmunoDiagnostics). In assays for antiviral IgA, wells coated with macaque IgA were used to generate a standard curve. Plates were developed with biotinylated, purified goat anti-human IgG Ab (Southern Biotechnology Associates, Birmingham, AL) or unfractionated goat serum IgG containing anti-monkey IgA Ab (Accurate, Westbury, NY) that had been depleted of Ab cross-reactive with monkey IgG and biotinylated as described (23). Final development was with avidin peroxidase and ABTS (Sigma-Aldrich, St. Louis, MO), followed by measurement of absorbance at 414 nm.

The end-point titer of anti-SIV or HIV EnvMN gp120MN IgG Ab was determined as the last serial 2-fold dilution of plasma that produced an absorbance value that was greater than the mean absorbance plus 3 SDs of eight blank (buffer diluent) wells that were reacted with the same reagents. Concentrations of SIV-, gp120-, or gp41-specific IgA were interpolated from standard curves constructed with the SoftMax Pro computer software program (Molecular Devices, Sunnyvale, CA). Antiviral IgA concentrations were divided by the total IgA concentration in each rectal secretion to obtain specific activity (nanograms of Ab/micrograms of Ig), which correlates directly with the proportion of virus-specific Ab-secreting cells in the rectal mucosa. Using the above assays, we have previously established that Ab titers or specific activity in postvaccination plasma or secretions are significant if they are 3.4-fold greater than those in corresponding preimmune specimens (23).

Measurement of neutralizing Abs

Neutralization titers were evaluated by an inhibition of infectivity assay (44). The virus used in the neutralization assay was SHIV89.6P (45). Ab-mediated neutralization of SHIV was be measured in an MT-2 cell-killing assay as described previously (46). Neutralization is measured at a time when virus-induced cell killing in virus control wells is >70% but <100%. Neutralizing Ab titers are given as the reciprocal dilution required to protect 50% of cells from virus-induced killing.

IFN-{gamma} ELISPOT

This assay was based on previously described methods (47) and followed the protocol provided by the manufacturer (monkey ELISPOT kit; U-CyTech, Utrecht, Netherlands). Briefly, 96-well microtiter plates were coated with 100 µl of anti-IFN-{gamma} Ab in PBS as instructed by the manufacturer and stored overnight at 4°C. After plates were washed and blocked, PBMC were plated in triplicate at two concentrations (105 and 5 x 105) with either medium alone (RPMI 1640 containing 25 mM HEPES, 10% FBS, L-glutamine, penicillin, and streptomycin) or medium containing 10 µg/ml Con A (Sigma-Aldrich), 5 µg/ml pooled SIV gag or SHIV89.6P env peptides (catalog nos. 6204 and 4827; AIDS Research and Reference Reagent Program), or an unrelated peptide pool. The plates were covered with a low evaporation lid and incubated at 37°C in 5% CO2 for 20 h. Following incubation, cells were flicked off, and residual cells were lysed with 200 µl of ice-cold water for 10 min. After washing the plates, spots were developed in accordance with the manufacturer’s instructions by consecutive treatments with biotinylated anti-IFN-{gamma} Ab, {gamma}-aminobutyric acid-conjugated anti-biotin Ab, and activator in substrate buffer. The spots in each well were counted by an independent automated immunospot image analyzer (Zellnet, New York, NY). The average number of spots present in the triplicate irrelevant cultures was subtracted from the average number in virus-specific peptide-stimulated cultures.

Intracellular staining for TNF-{alpha} and IL-2

Intracellular staining was conducted according to the previously described protocol (48). Briefly, 106 PBMC were stimulated with 1 µg/ml CD28 and CD49d Abs (BD Biosciences, Mountain View, CA) in the above-described RPMI 1640 medium and cultured for 1 h at 37°C in 5% CO2 with medium alone or medium containing 1 µg/ml staphylococcal enterotoxin B (Sigma-Aldrich) or 5 µg/ml pooled SIV gag and HIV Env peptides. After 1 h, brefeldin A (final, 10 µg/ml; Sigma-Aldrich) was added to each tube, and incubation was continued for 16 h. Cells were treated with 100 µl of ice-cold 20 mM EDTA for 15 min, washed with PBS containing 1% BSA, and then stained for 30 min on ice with anti-CD3-FITC (BD PharMingen, San Diego, CA) and antiCD4-PerCP or anti-CD8-PE (BD Biosciences). Cells were washed, permeabilized with FACS permeabilizing buffer (BD Biosciences), washed, and then stained with allophycocyanin-conjugated anti-TNF-{alpha} Ab or anti IL-2 Ab (BD PharMingen). Cells were fixed with 1% paraformaldehyde and analyzed for fluorescence by flow cytometry using a Beckman Cytomics FC500 (Beckman Coulter, Fullerton, CA). The CD3+ cells were used as the gate for CD8+ cells. Data for peptide-stimulated PBMC are reported as the percentage of TNF-{alpha}+CD3+CD8+ cells or IL-2+CD3+CD8+ cells, determined after subtracting the percentage of these cells observed after stimulation with an unrelated peptide pool.

Detection of Gag p11c-tetramer-staining CD3+CD8+ T lymphocytes

Rhesus macaques were screened for Mamu-A*01 positivity by a PCR-based technique (49). When the analysis was conducted on pooled mucosal biopsies, mononuclear cells (MNC) were isolated by collagenase digestion followed by Ficoll-Hypaque density gradient centrifugation. PBMC and MNC were stained with anti-human CD3 Abs (FITC-labeled; BD PharMingen), anti-human CD8{alpha}{beta} Ab (PerCP-labeled; Beckman Coulter), and a PE-labeled Mamu-A*01-Gag p11c conjugate (a gift from J. Altman (Emory University, Atlanta, GA)) (50, 51). The samples were analyzed by three-color flow cytometry on a Beckman Cytomics FC500, and the data are presented as percentage of tetramer-positive cells of all CD3+CD8+ cells. PBMC or mucosal MNC from two Mamu-A*01-negative animals were stained at every time point that was analyzed and the average of these results was subtracted from the values observed in Mamu-A*01-positive animals. To establish the cut-off for tetramer-positive responses, PBMC isolated from 10 nonvaccinated Mamu-A*01-positive and Mamu-A*01-negative animals were tested. The mean for gag p11c tetrameric complex staining background was 0.04%. A cut-off value of 0.1%, which is 2-fold higher than the mean background, was used as the threshold for positive responses in all of the PBMC tetramer data analysis.

Viral load quantitation

Plasma SHIV RNA levels were measured by a real-time RT-PCR assay, as described (52, 53). The assay has a threshold sensitivity of 100 copy equivalents per milliliter. Interassay variation is <25% (coefficient of variation).

Analysis of blood-derived immune cells

Characterization of CD4+ and CD8+ T cells in PBMC was conducted according to previously published procedures (54).

Statistical analysis

Calculations and statistical analyses were performed using the Statview 4 computer program (Abacus Concepts, Berkeley, CA). End-point Ab titers and RNA viral loads were logarithmically transformed, and the geometric means were calculated for each vaccination group. Between-group comparisons were performed by ANOVA using Fisher’s protected least significant difference set to the 95% confidence level. Within-group comparisons were by two-tailed, paired t test. Correlation analyses were done by Spearman rank followed by Fisher’s r-to-z conversion of correlation coefficients to p values. Results of statistical analyses were considered significant if they produced values of p <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our working hypothesis is that virus-specific mucosal responses offer a first line of defense that may eliminate or substantially reduce the virus inoculum, and that any eventual residual viral infectivity is controlled by systemic responses. Therefore, our goal is to develop a vaccination regimen that leads to sustained mucosal and systemic virus-specific responses. In the protocol reported here, we evaluated the immunomodulatory effect of IL-2 and IL-12 given as DNA vaccines on virus-specific mucosal and systemic antiviral immune responses when administered nasally.

Fifteen rhesus macaques were vaccinated nasally with SHIV DNA pVacc4 in saline solution. Group 1 received 1 mg of pVacc4 plus 0.5 mg of pUC19 DNA; group 2 received 1 mg of pVacc4 plus 0.5 mg of IL-2/Ig DNA; and group 3 received 1 mg of pVacc4 plus 0.5 mg of IL-12. The IL-2/Ig plasmid DNA construct expressed IL-2 as a fusion protein with the IgG Fc fragment (37); IL-12 plasmid DNA expressed macaque IL-12 as a single chain in which the two subunits p35 and p40 are linked by a 6-aa polypeptide linker. The animals received three DNA vaccinations, at day 1, wk 9, and wk 25, and an additional nasal vaccination with 109 PFU of MVA, expressing both SIV Gag-Pol and HIV Env 89.6P, on wk 33 to animals in all groups. The development of virus-specific immune responses was followed during the immunization, and the data are reported below.

SHIV-specific systemic and mucosal humoral immunity

Blood samples, rectal secretions, and saliva were harvested during the course of the immunization, and SHIV-specific IgA and IgG were measured in all of these samples. Humoral responses were mainly limited to SHIV Gag- and Pol-specific IgA in the rectal secretion of 10 animals (Table I). Three animals, two in group 1 and one in group 2, had anti-HIV gp120 IgA in rectal secretions. Only one animal in group 1 had anti-HIV gp41 IgA in the rectal secretions. No significant increase in salivary IgA Abs specific for SIV or the HIV envelope proteins was detected in monkeys at any time after vaccination. Significant, although very moderate, increases in anti-SIV lysate IgG compared with preimmune sera were found in the sera of animals 401, 397, and 409 (Table II). We could not detect boosting of humoral responses in the serum or in rectal secretions after the nasal MVA vaccination, and antiviral IgA had declined in most of the animals by the day of challenge, and only animals 399 and 400 retained low levels of anti-gp120 IgA on that day. A boosting of both virus-specific IgA in rectal secretions and IgG in the serum was observed in a previous vaccination protocol, when DNA and MVA were both administered rectally (our unpublished observations).


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  		Table I. SHIV IgA in rectal secretionsa

 

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  		Table II. SHIV-specific IgG Ab titers in plasmaa

 
These data indicate that a SHIV DNA/MVA vaccine administered nasally can stimulate rectal antiviral IgA in the majority of the animals but is not effective at inducing antiviral systemic IgG. IL-2/Ig or IL-12 DNA and the rMVA added to the vaccination did not result in significant differences in these humoral immune responses.

SHIV-specific systemic and mucosal cell-mediated responses

An expanding body of evidence supports the idea that virus-specific CD8+ cells, which can have CTL activity, play a major role in controlling viral replication in vivo and are likely to play a similarly important role in mediating protective immunity against infection with HIV (3, 35, 55, 56, 57, 58, 59, 60). IFN-{gamma} ELISPOT with PBMC, intracellular cytokine staining (ICS) for TNF-{alpha} and IL-2, and p11C tetramer staining of Mamu-A*01-positive macaque PBMC and of MNC from rectal mucosal biopsies were conducted to evaluate virus-specific cell-mediated immune responses. Responses against SIV Gag and HIV Env determinants were present in PBMC after the third DNA vaccination, which occurred on wk 25 (Figs. 1–3). The number of IFN-{gamma}-secreting cells was particularly high after HIV Env peptide stimulation of PBMC obtained from all vaccinated animals after the third DNA vaccination; however, these responses declined by the day of challenge. On the day of challenge, a larger fraction of the animals in group 2 had better Gag-specific responses than those observed in animals of groups 1 and 3 by ELISPOT (Fig. 1). Nasal administration of MVA resulted in a moderate boosting of systemic cell-mediated responses, in particular for the Env Ag when responses were analyzed by TNF-{alpha} ICS (Fig. 2, d–f). No major differences in systemic cell-mediated responses measured by TNF-{alpha} ICS were observed on wk 41 (day of challenge) between groups 1 and 3 (Fig. 2, d–f). Virus-specific cell-mediated mucosal responses, measured by evaluating the percentage of CD3+p11C+ cells (Fig. 3c, {blacksquare}) in rectal mucosal MNC of Mamu-A*01-positive macaques on wk 27, after the third DNA vaccination, indicated that these responses were generally higher than the virus-specific responses detected by tetramer in the PBMC (

) at the same time point. It is possible that the decrease observed for CD3+p11C+ PBMC on wk 27 compared with wk 26 resulted from the redistribution of these cells from the peripheral blood to the mucosal tissues (Fig. 3a).



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  		FIGURE 1. SHIV-specific cell-mediated systemic responses. a—c, Number of IFN-{gamma}-secreting PBMC detected by ELISPOT after stimulation with SIV Gag peptide pools. d—e, Number of IFN-{gamma}-secreting PBMC detected by ELISPOT after stimulation with HIV Env peptide pools. The dotted line at 50 spot-forming units corresponds to the level of 2-fold the average background and indicates the threshold for significant responses. Graph bars represent the mean number of spots per million cells detected in triplicate cultures of PBMC cultured with peptides, after subtracting the mean number of spots found in triplicate control cultures of PBMC in medium alone. Error bars represent SEM.

 


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  		FIGURE 2. SHIV-specific cell-mediated CD8+ T cell responses evaluated by TNF-{alpha} ICS. a—c, Number of CD8+, TNF-{alpha}-expressing T cells in PBMC after SIV Gag peptide pool stimulation. d—f, Number of CD8+, TNF-{alpha}-expressing T cells in PBMC after HIV Env peptide pool stimulation. The percentage shown represents data obtained after subtraction of the CD8+, intracellular TNF-{alpha}+ PBMC in control cultures. The dotted line at 1.2% corresponds to the level of 2-fold the average background and indicates the threshold for significant responses.

 


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  		FIGURE 3. Gag-specific p11C tetramer staining in PBMC and rectal mucosa MNC. a, CD3+CD8+p11c+ T cells in PBMC of Mamu-A*01 animals throughout the vaccination protocol. b, Flow cytometry analysis of CD3+p11c+ T cells in rectal biopsy MNC collected on wk 27 from one representative Mamu-A*01-negative and the six Mamu-A*01-positive animals in the study. c, The two graph bars for each Mamu-A*01 animal represent the percentage of rectal MNC CD3+ ({blacksquare}) and of PBMC CD3+ () T cells colabeled with Gag p11C tetramer detected on wk 27, after the third DNA vaccination.

 
These data indicate that both mucosal and systemic antiviral cell-mediated immunity was stimulated by the nasal vaccination in at least a subset of immunized animals. The administration of IL-2/Ig DNA to group 2 animals stimulated more significant and persistent anti-Gag cell-mediated responses.

Protection from SHIV infection and/or simian AIDS after mucosal challenge

Despite inconsistent systemic cell-mediated immune responses among the vaccinated animals after MVA boosting, we opted not to add additional vaccinations, because the immunization regimen would become overly complex and impractical for human vaccination. The 15 vaccinated animals and 5 naive controls were challenged rectally at wk 41, 8 wk after the MVA vaccination, when responses had declined to memory levels. Because the vaccine used for immunization in our study was a plasmid carrying a SHIV mutated provirus, animals in all groups were challenged with cloned SHIV89.6P, administered to the rectal mucosa. This amount of virus corresponds to ~10 50% animal infectious doses, titrated via the rectal route. An identical dose of the same virus stock had been used for rectal challenge in 20 additional animals, all of which became infected (our unpublished observations).

Animals were bled weekly to assay for the presence of virus in peripheral blood and to determine whether an anamnestic response to SHIV Ags was stimulated by exposure to the virus. RT-PCR was conducted to detect RNA viral loads in serum samples obtained on the day of challenge and up to wk 30 after challenge. Eighteen animals became persistently infected after the challenge (Fig. 4). Two Mamu-A*01 in group 1, which remain RT-PCR negative for the entire follow up, were transiently infected, as indicated by the significant increase in anti-gp120 Ab titer observed 6–8 wk after challenge (Table III). The difference in average viremia during the entire postchallenge time course between groups 1 and 3, groups 1 and 4, groups 2 and 3, and groups 2 and 4 was significant (p < 0.05). This statistical difference was even more striking when the analysis was restricted to the chronic phase of viremia (starting from wk 45, 4 wk after challenge and thereafter). There was no statistically significant difference when the mean viremia of group 1 was compared with group 2, but the comparison of these two groups was complicated by the fact that two animals in group 1 never became viremic, as measured by RT-PCR. Values reported in summary figures for RNA viral copy number (Fig. 4b) included all observed values, including the negative values that follow earlier positive time points in some of the animals and the persistently negative values observed in the PBMC of the two transiently infected animals.



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  		FIGURE 4. RNA viral loads in macaques challenged rectally with SHIV89.6P (expressed as log10). a, The value of viral RNA copies detected in the plasma of each animal is reported for individual time points. b, The geometric mean of viral RNA copy numbers detected in the animals of each group at each time point is reported. Error bars represent SEM.

 

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  		Table III. SHIV Abs in rectal secretions and in plasma of postchallenge RNA (-) animalsa

 
Seroconversion could be documented for all of the animals by an increase in SHIV-specific serum IgG in infected animals, beginning 2–3 wk after challenge (Fig. 5). The kinetics and magnitude of the antiviral IgG Ab response during the first 4 wk after challenge were similar in naive controls and in most of the vaccinated animals, suggesting that the virus-specific Abs detected in the animals after challenge were a primary, rather than secondary response against viral Ags. However, 6–8 wk after challenge, differences in the Ab response were observed. At this time, group 2 animals were found to have significantly greater titers of HIV gp120- and gp41-specific IgG than group 1, 3, and 4 animals (p < 0.04 for all comparisons by ANOVA). By 12–16 wk after challenge, group 2 macaques had also developed significantly greater titers of SIV-specific IgG in plasma than those in groups 1, 3, and 4 (p < 0.05 for all). The magnitude and the persistence of serum IgG responses detected up to 20 wk after challenge in the chronically infected animals correlated directly with the levels of CD4+ T cell count and inversely with the level of the viremia. This result suggests that containment of virus replication and consequent preservation of the immune system is critical to the establishment of significant Ab responses.



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  		FIGURE 5. Postchallenge systemic Ab responses to SHIV. Geometric mean reciprocal end-point titers of anti-SIV Gag/Pol- (a), anti-HIV gp120 (b), anti-HIV gp41-specific IgG (c), and neutralizing Abs (d) measured in plasma collected on the day of challenge (wk 41) and at intervals up to 20 wk later (wk 61). Error bars represent SEM.

 
Neutralizing Abs against SHIV89.6P were also measured in serum from the animals after challenge (44). Neutralization titers were negative on the day of challenge and 4 wk after challenge, became detectable by wk 8, and increased with time in some of the animals. The similar timing of the development of this response in group 1, 2, and 3 vaccinated animals compared with group 4 and historic controls excludes priming of neutralizing Abs by the vaccination regimen (61, 62, 63, 64). After wk 45, titers of neutralizing Ab increased in only a few animals of groups 1, 3, and 4. In contrast, these titers continued to increase in all group 2 animals, and at wk 61, were significantly greater than those in groups 1, 3, and 4 (p < 0.008, p < 0.008, and p < 0.02, respectively) (Fig. 5d). There was a direct correlation between the presence of neutralizing Abs, the control of viremia, and CD4+ T cell counts. It is possible that the preservation of CD4+ T cells due to the vaccine-induced cell-mediated responses facilitates the development of Ab responses in general, including neutralizing Abs, and these in turn contribute to a reduction of viremia and protection of the immune system.

The two animals (395 and 398 of group 1) in which viremia was never detected by RT-PCR during the follow-up had significant serum IgG seroconversion against gp120 (5- and 8-fold increases over the day of challenge titer), and a 6.6-fold increase in anti-gp120 IgA in rectal secretions (animal 398) 6 wk after challenge (Table III). No significant increase in Ab titer against the total SIV lysate (which measures almost exclusively Ab against SIV Gag and Pol) was observed in these two animals. It is important to note that the Env responses are measured with an ELISA plate coated with HIV Env MN and not Env 89.6P, which is the Env present in the SHIV DNA used for immunization and in the challenge virus, and therefore, these responses could be underestimated. The anti-gp120 responses declined below the level of significance after wk 8 postchallenge in animal 395 and remained significant for the entire follow-up, although lower than the peak, which was reached at wk 6 for 398. Considering the prolonged life of circulating Abs, these results suggest a reduction of antigenic stimulation and possibly viral clearance.

CD4+ T cell counts were evaluated in PBMC as a surrogate marker of disease progression (Fig. 6). As previously observed in HIV-infected individuals and SIV- or SHIV-infected animals, the levels of CD4+ T cell counts inversely correlated with SHIV RNA viral loads (63, 65). The difference in the CD4+ T cell counts observed during the entire follow-up between groups 2 and 3 and between group 2 and the controls in group 4 was statistically significant (p < 0.05). Five of five group 2 animals, which received IL-2/Ig DNA in addition to the SHIV DNA, remained alive and well up to 43 wk postchallenge, when they were euthanized because of termination of the protocol. During the time that preceded this date, four of the five group 4 control animals, one of group 1, and four of group 3 had been euthanized, because they had developed AIDS.



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  		FIGURE 6. Postchallenge absolute CD4+ T cell counts per milliliter (a–e) and CD4+ T cell percentage (f) in PBMC of animals in the three vaccine groups. CD4+ T cells were evaluated in PBMC on the day of challenge (wk 41) and up to 25 wk postchallenge (wk 66). e, The mean of the CD4 T cell counts detected in PBMC of the animals of each regimen group is reported. f, The mean percentage of the CD4+ T cells detected in PBMC of the animals in each regimen group is reported. Error bars represent SEM.

 
Although the role of CD8+ T cell responses in viremia control has been investigated intensively, the characterization of CD4+ T cell-mediated responses during chronic infection has received less attention. Recently, McKay et al. (66) showed that preservation of virus-specific functional CD4+ T cells correlates with control of viremia. Production of cytokines by CD4+ T cells plays a major role in coordinating humoral and cellular responses against infectious agents and in determining the outcome of infection (67). Investigation of CD4+ helper responses in SIV{Delta}nef-immunized animals showed that stimulation of these cells with SIV p55-related peptides resulted in induction of IFN-{gamma} and IL-2 but not IL-4 and IL-10 (22). We analyzed the Th virus-specific responses by evaluating CD4+ T cells that express IL-2 and TNF-{alpha} after virus-specific stimulation on the day of challenge and 8 wk later (Fig. 7a). Gag-specific tetramer staining performed postchallenge in PBMC did not reveal any specific trend (Fig. 7b). Virus-specific CD4+ T cells were close to background levels in most of the vaccinated animals on the day of challenge. Higher levels of virus-specific CD4+IL-2+ T cells were observed in group 2 animals 8 wk after infection (wk 49), confirming the correlation between CD4+ T cell responses and viremia control described by McKay et al. (66). Interestingly, a higher percentage of ICS for IL-2 than for TNF-{alpha} was observed after SHIV peptide stimulation in CD4+ T cells of group 2 animals, which had a better prognosis. The opposite was true in CD4+ T cells from animals in the other groups. An increase in anti-Gag and anti-Env CD4+ T cell responses over the levels observed on the day of challenge could also be detected in the two animals of group 1 that were transiently infected. Preservation of both systemic and mucosal virus-specific CD4+ T cell responses (measured as ICS for IL-2 and IFN-{gamma} in CD4+ T cells) was observed in all disease-free animals that were analyzed 43 wk after challenge, when they were euthanized, as the study was closed (Table IV).



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  		FIGURE 7. Cell-mediated CD4+ T cell responses after challenge. a, Number of CD3+CD4+ TNF-{alpha}- and IL-2-expressing T cells in PBMC after SIV Gag or HIV Env peptide pool-specific stimulation. Challenge occurred on wk 41. Error bars represent SEM. b, Gag-specific tetramer staining in PBMC after challenge. The average percentage of values of CD3+CD8+ T cells labeled with Gag p11C tetramer detected in PBMC of Mamu-A*01 animals present in each group is represented.

 

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  		Table IV. Systemic and mucosal virus-specific cell-mediated responses in selected surviving animals 43 wk after challengea

 
Taken together, these data suggest that the nasal vaccination with SHIV-DNA and IL-2/Ig DNA plus rMVA provided long-lasting protection from disease progression, and that the presence of SHIV Gag-specific responses on the day of challenge correlated with normal CD4+ T counts and protection from AIDS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We reported a preclinical vaccine trial aimed at evaluating whether nasal vaccination can stimulate both anti-SHIV mucosal and systemic immunity. We found that a DNA-MVA regimen that included IL-2/Ig DNA administered nasally can induce antiviral mucosal and systemic immune responses and can provide protection from AIDS much like some vaccines administered parenterally (63, 65). It may be important to have virus-specific mucosal immunity in protection against HIV. It has been suggested that secreted Abs might provide the first line of defense against the virus inoculum transmitted at the time of exposure, and local interstitial Abs and CTLs could act as the second line of defense against virus that enters the mucosa (68, 69). Regional immunity at iliac lymph nodes could eliminate any residual infectivity that is not controlled at the mucosal membranes. Immunologically mediated containment of the infection during its initial local phase might be possible even in the absence of sterilizing immunity, and the presence of mucosal immune responses could be critical to achieve this goal. However, once chronic viremia is established, clearance of infection appears to be extremely difficult, even when the infection is due to highly attenuated viruses (70, 71). If immunologically mediated viral clearance cannot be achieved, control of virus replication and disease delay become necessary features of vaccines aimed at providing protection from AIDS.

The cell-mediated mucosal and systemic immune responses induced by the vaccination in group 2 correlated with delaying or preventing CD4+ T cell loss in some of the animals. However, because other immunological parameters potentially relevant to viremia control and disease protection were not extensively evaluated in this study (in particular, mucosally located cell-mediated responses), it is difficult to derive the full set of correlates of protection. Despite the small group size and the variation of the outcome in the animals in three of the four groups, significance in the postchallenge outcome was observed when the outcome of groups 1 or 2 was compared with groups 3 and 4.

There are a number of possible explanations for the results we observed. Anti-Gag cell-mediated responses present on the day of challenge in group 2 animals and detected by IFN-{gamma} ELISPOT appeared to correlate with the viremia control observed in this group and could account for the protection from disease. The substantial increase of SHIV-specific IgA responses observed in at least three of the control animals soon after challenge appears to correlate with the high viremia present in these animals and could reflect significant virus replication at mucosal sites. The quick decline of this response in the same animals parallels the decline in CD4+ T cell count and probably reflects the failing of the immune system. Limited and sporadic development of significant IgA responses after challenge in the group 2 animals could reflect controlled replication in the intestinal mucosa mediated by antiviral cell-mediated responses that were not fully evaluated in this study. Differences in virus-specific Th cell responses could account for some of the observed postchallenge differences as well. The lack of correlation between the pre-existing antiviral immune responses and the outcome of group 1 and 3, for which mixed results were observed, might depend on the small sample size of each group. However, it is unlikely that the prolonged protection of five animals of five in group 2 is a random result. This rate of protection is comparable with the rate of success obtained by others, using SHIV DNA vaccination with IL-2/Ig DNA via the systemic route (63).

Two animals in the experiment described here achieved contained infection and may have been protected by immune responses induced through vaccination (animals 395 and 398 of group 1). However, as transient infection has been observed in mucosally exposed nonvaccinated animals (72, 73, 74), we cannot establish whether or not the clearance of infection depended strictly upon the immune responses induced by the vaccination. It is important to note that transient infection and clearance have usually been achieved with very small doses of virus (72, 73, 74), and this was not the case in our experiment.

Because the only indication of infection in animals 395 and 398 is the variation in Ab titer, it could be argued that this parameter is not sufficient to indicate infection and might simply reflect exposure to viral proteins. It is highly unlikely that the increase in Ab titer results simply from exposure to the viral proteins present in the challenge virus, because the amount of virus-associated p27 present in the challenge dose was ~10 ng, and the amount of Env is likely to be less (ratio of Gag:Env, ~10:1). This amount of protein is too small to stimulate an increase in Ab titer, in particular if given in absence of adjuvant. It is more likely that containment of infection occurred in these animals, and the short-lived infection was responsible for the small Ab titer increase and subsequent decline and for the expansion of virus-specific CD4+ T cell responses. Interestingly, these two animals had very strong p11C-specific cell-mediated mucosal responses, as measured by tetramer staining, in lymphocytes derived from the rectal mucosa (Fig. 1c). These pre-existing responses could have mediated the mucosal containment of the infection.

We could not conclusively evaluate the adjuvant role of IL-12 in stimulation of virus-specific responses, because it is not clear whether the observed results were affected by group size, in particular when the results for group 3 are compared with those of group 1, in which no adjuvant cytokine was administered, and two animals did not become persistently infected. Additional experiments are needed to fully evaluate the adjuvancy of this cytokine. Instead, this vaccine trial provides evidence for a beneficial role of IL-2 used as adjuvant on immune responses in mucosal vaccination. The systemic responses achieved in group 2 with mucosal vaccination provided control of viremia to a degree similar to that achieved with i.m. vaccination in the same SHIV89.6P model (37, 63). The observed results may not apply to viruses different from SHIV, such as HIV or SIV, which have a less acute impact on the immune system and are more difficult to control immunologically (75). Furthermore, the validity of the nasal route of vaccination in the SIV model needs to be addressed. Nasal vaccination with attenuated NYVAC/SIVgpe stimulated significant antiviral responses in two female macaques, both systemically and at different mucosal sites (21). However, the number of animals in the group and the follow-up after challenge for only 48 h prevent the generalization of the results.

It is our opinion that mucosal challenges in animal models will not be able to fully address the significance of mucosal immunity in infection control at the site of exposure and prevention of chronic infection. The level of virus in the inoculum used at the time of mucosal challenge (109 viral RNA copies), which is needed to achieve 100% infection in control animals, is significantly higher than the median RNA copy number observed in the semen of HIV-infected men, which likely corresponds to what is transmitted during natural HIV exposure (76, 77, 78, 79). It is unlikely that any vaccine can stimulate immune responses that can contain locally this amount of challenge virus. However, the same level of mucosal response may contain the more limited amount of virus present in the inoculum transmitted during natural exposure. With these issues in mind, an experimental mucosal challenge, as the i.v. challenge, can only evaluate whether the vaccine-induced virus-specific systemic immunity is effective at containing systemic chronic infection.

The role of virus-specific mucosal responses in prevention or local containment of infection will most likely be evaluated only in human trials. However, it is important to develop animal vaccination regimens that can stimulate virus-specific mucosal immunity in addition to systemic immune responses, because these responses might be sufficient to control the infection locally when present in exposed humans. This possibility is less likely after parenteral vaccination, which is not as efficient as mucosal vaccination in stimulating mucosal responses. A future goal should be to devise immunization regimens that are capable of stimulating high levels of both mucosal and systemic immunity and yet are practical for clinical testing. The fact that significant levels of systemic and mucosal immune responses and protection from disease were achieved in our study provide a strong rationale for expanding this approach and evaluating which vaccination regimen can provide the most diverse virus-specific immune responses.


   Acknowledgments
 
We thank Drs. Norman Letvin and Dan Barouch for providing the IL-2/Ig plasmid, Dr. Richard Mulligan for the human IL-12 expression plasmid, and Dr. John Altman for the gift of MHC tetramers. We also thank Dr. Jeff Lifson and Mike Piatak for assistance with the measurements of viral loads in macaques.


   Footnotes
 
1 This project was funded by National Institutes of Health Grants AI41365 (to A.A.) and AI48133 (to P.A.K.) and was supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-124000. Back

2 Address correspondence and reprint requests to Dr. Anna Aldovini, Department of Medicine, Children’s Hospital, Enders 609, 300 Longwood Avenue, Boston, MA 02115. E-mail address: anna.aldovini{at}childrens.harvard.edu Back

3 Abbreviations used in this paper: SHIV, simian/human immunodeficiency virus; MVA, modified vaccinia virus Ankara; MNC, mononuclear cell; ICS, intracellular cytokine staining. Back

Received for publication August 4, 2003. Accepted for publication January 7, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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