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Journal of Virology, April 2008, p. 3166-3180, Vol. 82, No. 7
0022-538X/08/$08.00+0     doi:10.1128/JVI.01634-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Human Immunodeficiency Virus Type 1 Vaccine Development: Recent Advances in the Cytotoxic T-Lymphocyte Platform "Spotty Business"

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

	INTRODUCTION

Top INTRODUCTION REFERENCES

 
For human immunodeficiency virus type 1 (HIV-1) vaccine development, a central focus is developing a vaccine that primarily drives strong and broad CD8⁺ cytotoxic T lymphocytes (CTLs). This focus is largely due to the difficulty in generating cross-neutralizing antibodies against the diverse array of HIV viral envelopes (9, 40, 78, 139). The ability of CD8⁺ T cells to impact or control viral replication is supported by model primate studies and is reiterated by studies of immune control in infected individuals. For example, Cao et al. (28) reported that HIV-1-infected long-term nonprogressors exhibited high levels of HIV-specific CTLs. In addition, HIV-1-infected patients with high levels of virus-specific CTLs exhibited lower viral loads, slower CD4⁺ T-cell declines as measured in peripheral blood, and relatively stable clinical statuses (23, 81, 108, 112). Preclinical studies conducted with simian immunodeficiency virus (SIV)-infected monkeys showed that upon their in vivo depletion of CD8⁺ T cells, a rapid and dramatic increase in viremia was observed (70, 131). While no correlates of immunity for protection against HIV-1 have been concretely established, these data as well as those of others (6, 13, 137) strongly suggest that CD8⁺ T cells are important in controlling viral infection and may be an important component of an effective HIV-1 vaccine. In this review, we summarize recent advances in HIV vaccine development utilizing various platforms that target the induction of cellular immunity. In particular, we draw inferences, as appropriate, for the induction of cellular immune responses at mucosal as well as systemic sites.

	CTL-BASED VACCINES: STEPPED ON?

 
The discontinuation of the Merck recombinant adenovirus type 5 (rAd5) phase II proof-of-concept STEP study is undoubtedly a significant setback for the field of HIV vaccine development. In the study, a trivalent, three-vector rAd5 (Gag, Pol, Nef) vaccine was tested in two cohorts of 1,500 individuals at high risk for HIV infection. The vaccine was scheduled for three immunizations of 1.5 x 10¹⁰ viral genomes of each rAd5 vector encoding Gag, Pol, or Nef administered on day 1, month 1, and month 6. There are concerns that preexisting vector serology can mute the effectiveness of a CTL vaccine approach. Accordingly, the first cohort was made up of 1,500 individuals with low levels of preexisting antibodies against Ad5, with titers of 1:200, while the second cohort of 1,500 included participants with anti-Ad5 titers of greater than 1:200. However, the STEP study was prematurely stopped by the data- and safety-monitoring board because it was clear that even in the cohort with low titers of preexisting antibodies against Ad5, there was no evidence of efficacy. Table 1 summarizes the results from the cohort with titers of 1:200, comparing the rates of infection and viral loads of the vaccinated versus placebo groups. This was the best-response group in earlier clinical studies, eliciting CTL responses in over 60% of vaccinees. As shown in Table 1, in the subset of individuals that received at least two immunizations, there was a trend toward more HIV infections in the rAd5-vaccinated (19/672) group than in those in the placebo (11/691) group. The difference in viral loads 12 to 18 weeks postacquisition of the infection between the vaccinated and placebo groups (40,000 versus 37,000 copies per ml, respectively) were not statistically significant. Since this rAd5 trivalent vaccine did not prevent infection or decrease viral replication in vaccinated individuals, the implications for CTL-based vaccines remain unclear, except that future studies should test hypotheses and concepts that are not identical to the Merck approach. Furthermore, this study draws a line in the sand in that the following CTL-based approach should induce higher levels of CTL responses and induce a different immune phenotype. The inclusion of different antigens or prime-boost strategies in future studies with defined immune endpoints will be important. We will consider many of these in the following sections.

	THE IMPORTANCE OF MUCOSAL RESPONSES

View larger version (38K): [in this window] [in a new window]   FIG. 1. Vaccine induction of mucosal immunity may be important to prevent HIV-1 dissemination. HIV-1 is primarily transmitted through the mucosal surfaces. HIV-1-specific IgA antibodies could potentially neutralize the virus or block viral adherence or entry to mucosal tissues. CTLs specific for HIV-1 could lyse infected cells that present viral antigens via MHC-I, thereby preventing their entry into the systemic compartments. sIgA, secretory IgA.

	VIRAL VECTORS

Adenovirus. Adenovirus is a nonenveloped DNA virus that is typically acquired by humans in childhood. Clinical manifestations model the common cold and include respiratory, gastrointestinal, and ocular infections. While symptoms are transient, some serotypes are capable of persisting for much longer (months to years) in mucosal tissues and continuing to shed virus. Adenovirus has been studied extensively as a vaccine platform due to its high transduction efficiency, ease of manipulation, and ability to quickly grow to high viral titers in culture. In addition, recombinant adenoviral vectors can potentially carry large transgene inserts (up to 8 kb) when regions of the viral genome dispensable for viral growth (i.e., E1, E3) are deleted (21, 74). rAd5 is rendered replication deficient by deletion of the E1 region, which is essential for adenovirus replication as well as expression of downstream viral genes. The antigenic transgene is inserted in place of the E1 region under the control of a promiscuous promoter like cytomegalovirus and flanked by a bovine growth hormone polyadenylation terminus signal. Upon administration of a rAd5 vector plus an antigenic insert, the recombinant viral vector binds cells expressing the coxsackievirus and adenovirus receptor, thus facilitating its internalization. As a result, antigen synthesis occurs intracellularly, which leads to efficient processing and presentation of the antigen by MHC-I (reviewed in reference 11). A robust cellular and humoral immune response against the viral vector and transgene insert is observed, in part due to its intrinsic hexon protein-adjuvanting properities (104).

Of the 51 currently known serotypes, rAd5 has received the most attention for use as a vaccine vector and is currently in several clinical trials. However, the potent immune response generated against the Ad5 vector that makes it a promising HIV-1 vaccine candidate also creates one of the most difficult challenges for its development. The prevalence of preexisting anti-Ad5 immunity is astoundingly high among populations that are in desperate need of an HIV-1 vaccine, including many regions of sub-Saharan Africa (12, 80, 110, 158). In fact, Ad5 seroprevalence is greater than 90% in some regions of Africa, with much of the population exhibiting high titers of neutralizing antibody. Ad-specific immunity resulting from natural Ad5 infections decreases the immunogenicity generated against the antigen insert in the recombinant Ad5 vectors (143). In a phase I clinical trial carried out by Merck using rAd5-Gag, the vaccine elicited cellular immune responses in just 28% of subjects that had moderate levels of preexisting Ad5 neutralizing-antibody titers (>200), whereas Gag-specific cellular immune responses were observed in 82% of subjects with no Ad5 immunity (reviewed in reference 12). However, the trivalent vaccine was more effective at an increased dosage (36). Clearly, the significance of developing a vaccine to avoid preexisting antibody responses may be important, and studies using chemical alterations, chimeric Ad vectors, chimpanzee Ad vectors, and rare Ad serotypes found in humans are in development. Chemical modifications to aid Ad5 vectors in immune escape include the addition of microparticles (128) or polyethylene glycol (38) to reduce neutralizing-antibody recognition and increase the effectiveness of multiple immunizations. Another strategy is the construction of chimeric Ad vectors, which are created by swapping regions of the Ad5 fiber or hexon proteins with the corresponding regions of a different serotype. Chimeric Ad hexon vectors are capable of partially evading the antibody response against Ad5; however, they can be difficult to construct due to the preservation of low-titer neutralizing-antibody epitopes (50, 150), low-titer vector replication (126), and the induction of cross-reactive CTLs to other proteins of the virus (167). Barouch's group constructed a rAd5/Ad48 chimeric vector that replaces Ad5 neutralizing-antibody targets with Ad48 surface loops and has been shown to be immunogenic in rhesus macaques despite anti-Ad5 immunity (122). This important modified vector is moving forward into phase I clinical trials in collaboration with the International AIDS Vaccine Initiative (IAVI) (69). Novel rAd5 vectors are also being constructed using nonhuman Ads from ovine, porcine, bovine, and chimpanzee sources (45, 65, 119, 120, 154, 161). In preclinical trials, chimp vectors appear to be highly immunogenic, with T-cell responses against transgenes marginally affected by preexisting antibodies to human Ad5 (46, 117). In addition, chimp vectors elicit antibodies against the transgene in the presence of preexisting antibodies against human Ad5, whereas antibodies against the encoded transgene product are completely inhibited when delivered by the human Ad5 (100). Another strategy is the use of less-prominent adenovirus serotype antibodies found in humans, such as Ad6 (reviewed in reference 12), Ad35 (158), Ad11 (67), and Ad26 (1). Specifically, it has been reported that Ad35 is immunogenic and not inhibited by preexisting anti-Ad5 antibodies in mice (12); however, Ad35 elicits less-potent antitransgene responses than recombinant Ad5 vectors (11, 136). Ad35 is in clinical development by the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), and GenVec. Based upon the safety data for Ad35 from 15 volunteers, a second part of the trial will pursue the delivery of Ad35 in combination with Ad5 (69). Ad26 is another adenovirus serotype, less prevalent in humans, that will be moving into clinical trials (69). In rhesus macaques, rAd26 elicited strong cellular and humoral immune responses similar to those observed with rAd5 (1). In addition, an rAd26 prime-rAd5 boost regimen elicited cellular responses 10 times that observed with a homologous rAd5 immunization and partially controlled viremia following a SIVmac251 challenge (84). Recombinant adenovirus vectors are also attractive vaccines for HIV-1 because they can be administered at mucosal sites, both intranasally and orally. Furthermore, they have an intrinsic tropism for mucosal tissues, where they can persist for some time. In mice, adenoviral vectors have been shown to elicit cellular immune responses in the mucosa (92, 147); however, there is limited mucosal data on the use of these vectors in nonhuman primates, and it will be important to further characterize these responses.

	ONE STEP FORWARD: LESSONS LEARNED FROM NONHUMAN PRIMATE STUDIES

 
Studies comparing cellular responses induced by recombinant adenovirus, poxvirus, alphavirus, and plasmid DNA vaccines, all expressing SIV Gag in macaques, supported that rAd5 elicited the highest levels of antigen-specific CTLs, supporting its further clinical advance (136, 137). The results of studies with rAd5 in animal models highlight considerations for evaluation of candidate vaccines. The rAd5 vaccine never protected against infection: regardless of the monkey haplotype or challenge virus studied, all monkeys became infected following challenge, leading to the unrealistic expectation that vaccination with the rAd5 trivalent vaccine would protect against infection in humans. There was a clear hierarchy of control of viral replication by rAd5 vaccines in primate challenge models. In hindsight, the "easiest" challenge model, A01-positive (A01⁺) macaques challenged with simian-human immunodeficiency virus (SHIV) 89.6p, produced a clear 3-log reduction in viral loads compared to those of the control (137). Challenge with the more-virulent strain SIVmac239 resulted in a sustained 1.5-log decrease in the viral loads of rAd5-immunized A01⁺ monkeys (101). It is clear that Mamu A01⁺ rhesus macaques are useful models for observing antigen-specific responses by using tetramer staining, but it is questioned whether they are accurate models for predicting vaccine efficacy in humans. In preclinical studies with the rAd5 vaccine testing outbread macaques challenged with SHIV 89.6, a more-variable 2- to 3-log reduction was observed (89). However, when outbred macaques were challenged with SIVmac239 post-rAd5 immunization, no significant reduction in viral load was seen in vaccinated monkeys compared to that of the control (29). This hierarchy establishes that the use of outbred macaques and challenge with SIV should be considered the most-stringent vaccine model and be a focus for future studies evaluating HIV vaccine efficacy. However, based on a lack of evidence of protection competency in this model, it is clear that all primate studies are still at-risk studies without known correlates or clear immune targets and without a guarantee that protection in stringent SIV challenges guarantees similar protection in humans.

	ONE STEP BACK: IMPLICATIONS FOR CTL-BASED VACCINES

 
Merck, the HIV Vaccines Trial Network (HVTN), and the Division of AIDS, NIH, should be commended for their valiant effort, creativity, organization, and management of this important efficacy trial. The bar for the STEP study was set high, as rAd5 was the most-promising CTL platform at the time, based upon clinical and preclinical data for rhesus macaques. In earlier clinical trials, significant response rates to all three HIV antigens (73% to Gag, 59% to Pol, and 68% to Nef) among individuals with low seropositivity to Ad5 who were vaccinated three times with the trivalent Ad5 were observed at week 30. The reported geomean of responders as measured by gamma interferon (IFN-) enzyme-linked immunospot (ELISPOT) assays were 205, 342, and 177 spot-forming units (SFU) to Gag, Pol, and Nef, respectively (37). Response rates were lower at the same timepoint for Ad5-seropositive individuals with high virus titers, with 69% responding to Gag (406 SFU), 38% to Pol (834 SFU), and 54% to Nef (428 SFU) (37). The majority of responders responded to only one eptitope in the Gag, Pol, or Nef peptide pools, with approximately 30% of responders recognizing three or more epitopes (37). Although the trial consisted primarily of men who have sex with men, it is interesting that one woman in the placebo group was infected. This critically important observation highlights the importance of the clinic as a teaching ground for HIV vaccine development. This observation has implications for HIV transmission between men who have sex with men versus heterosexual transmission that must be considered in future trial development. With this in mind, the analysis of the responder data from the STEP trial will be critical in determining a relationship among vaccine responders, the levels of the response, and the resulting incidence of infection. In addition, we anxiously await the data analysis from the high-titer vector-seropositive cohort as well as the Phambilli study to see if these results reflect an increased trend for infectious compared to low-titer Ad5-seropositive cohorts. The increased trend toward infection is not likely a general trend for all vaccines that elicit T cells. The Vaxgen protein trial, which mobilized B and T helper cells, did not show a similar trend, nor did the ALVAC trials, which mobilized weak CD8⁺ T cells. It should be noted that this trend is being debated at this time, and therefore its importance to vaccines under development remains speculative.

Unanswered questions include the following. Are the response levels (on average about 400⁺ SFU) observed in responders high enough? Are the vaccine targets the most relevant? Would polyfunctional immune responses or some other T-cell phenotype change vaccine efficacy? Do the responses need to be directed against a broader number of epitopes to be effective? What animal models are most important as predictors of human vaccine efficacy? What do the mucosal responses in Ad5-vaccinated people look like, and would such responses change the impact of the vaccine? Why are the acquisition rates different between men and women?

	ADENO-ASSOCIATED VIRUS

 
Adeno-associated virus (AAV) is a helper-dependent parvovirus that has been well characterized as a gene therapy vector (reviewed in reference 144). The generation of the recombinant vector entails removing all viral genes except the sequences that encode the GC-rich inverted terminal repeats (ITRs). The entire genome is a mere 4.7 kb, which limits the size of the antigenic insert (between the ITRs) to roughly 3.8 kb, excluding the promoter and poly(A) tail. The removal of viral genes reduces the intrinsic immunogenicity of AAV; however, rAAV remains an attractive vaccine candidate due to the ability to produce it on a commercial scale (34), its outstanding clinical safety profile for nearly a decade (reviewed in reference 72), the ease with which it can be administered intramuscularly, and the high transduction efficiency of myofibers (reviewed in reference 72). Johnson et al. (72) demonstrated the efficacy of rAAV for delivery of SIV antigens (SIVsm/E660) in an intravenous challenge model. A portion of their study included two injections of three rAAV vectors encoding SIV genes at doses measured by DNase-resistant particles, which are equivalent to encapsulated vector genomes. The three rAAV vectors contained fragments of (i) SIV Gag, protease, and reverse transcriptase; (ii) SIV Rev and Env; and (iii) reverse transcriptase and integrase at 10¹³, 5 x 10¹², and 10¹³ DNase-resistant particles, respectively. The results showed robust cellular (tetramer-specific CTLs) and humoral (serum antibody) immune responses after a single injection. In fact, after a virulent intravenous challenge, immunized monkeys exhibited significant control of SIV replication, with viral loads almost 2 logs lower than those of the control group. The protection from challenge may be attributed to CTLs, as there were no detectable neutralizing antibodies against the SIV challenge stock observed in the sera of vaccinated animals. Interestingly, there was no increase in the observed immune response after the second immunization, suggesting the generation of antivector antibodies. In fact, the preexisting or vaccine vector-induced immunity to the capsid is still a major concern for this platform. For example, recombinant AAV type 2 (AAV2) is the most-studied serotype to date, and it has been shown that 27% of the population has neutralizing antibodies directed against AAV2 (162). Similar to strategies being employed with recombinant adenoviral vectors, less-prevalent rAAV serotypes found naturally in humans, including AAV1 and AAV5, are being examined to circumvent this issue. Alternating rAAV capsid serotypes from one immunization to the next is being explored. A phase II clinical trial organized by Targeted Genetics and IAVI utilizing rAAV encoding HIV Gag, Pol, and reverse transcriptase (tgAAC09) is under way in Zambia and has been reported safe and well tolerated thus far. While initial clinical studies using rAAV appear to be overwhelmingly safe, recently a patient participating in a phase I/II gene therapy trial for the treatment of rheumatoid arthritis died after receiving a rAAV vector encoding the soluble tumor necrosis factor alpha receptor tgAAC94. The patient was also taking a tumor necrosis factor alpha blocker, Humira, which is immune suppressive. While there has been no report of a definitive determination of the cause of the patient's death, it appears likely that infection with a fungus, Histoplasma capsulatum, could be the cause (75). The role of the AAV treatment in this patient's outcome is not clear. Importantly, this study highlights the potential pitfalls of performing trials in immunosuppressed individuals whose responses are clearly different from those of immunocompetent people. The immunogenicity of rAAV vectors clearly needs improvment, and the use of molecular adjuvants and prime-boost strategies will undoubtedly be tested and will be discussed in future sections. The published data on the generation of mucosal immune responses by rAAV is limited, although it is an interesting vector due to its stability at a wide range of pH values and high temperatures.

	ALPHAVIRUSES AND RHABDOVIRUSES

 
Alphaviruses are positive-stranded RNA viruses with a broad host range that cause a variety of mosquito-transmitted diseases, including Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis (VEE), in the United States. While the majority of alphaviral infections are asymptomatic in humans, flu-like symptoms, including fever, headaches, and body aches, can occur. The virus is also capable of invading the central nervous system, leading to encephalitis and perhaps even death. Modifications to the virus for the generation of vaccine vectors has been well studied (reviewed in references 118 and 130). To generate vaccine replicon particles, the nonstructural protein genes and natural subgenomic promoter (26S) are retained, while the structural protein genes are replaced with the antigenic gene of interest. Current alphavirus replicon particle vaccines have been derived from the Sindbis virus, Semliki Forest virus (91), and VEE virus. This process creates a single-cycle replication vector that is translated upon entry into the cytoplasm of the target cell, allowing for intracellular processing and subsequent efficient presentation by MHC-I. The cytoplasmic amplification of these vectors includes a double-stranded RNA intermediate that activates both the innate and adaptive immune systems, strengthening this platform as a vaccine candidate delivery system (85). Indeed, the replication of alphavirus replicons results in extremely high levels of antigen expression (157, 166, 171); however, this replication is generally toxic to the infected cell, thus preventing long-term antigen expression (48). The antivector immune response against alphavirus replicons has so far been minimal, allowing for readministration, a major benefit for a vaccine (118). Similarly, seroprevalence is low in humans, and the replicons can be engineered to be lymph node trophic, enhancing antigen presentation through efficient targeting to, and expression in, the immune inductive site. A possible drawback to the alphavirus platform includes the requirement of the Rep protein for high-level antigen expression. This antigen may be prone to immune surveillance by CTLs. However, this platform has many positive features. For example, previous studies report that the primary cellular targets of replicon particles have been shown to be those of the CD14⁺ monocyte lineage in rhesus macaques (55) and dendritic cells in human peripheral blood mononuclear cells (51). In nonhuman primates, vaccination with 2 x 10⁸ PFU of chimeric SINenv and VEErep alphavirus replicon particles expressing SIVp55Gag and/or HIVV2gp140 alone resulted in high titers of anti-HIV neutralizing antibodies, while moderate anti-Env IFN- ELISPOT responses were also observed (163). In a separate study, 1 x 10⁷ infectious units of each of three VEE virus replicon particles expressing the SIVsmH-4 matrix capsid region of Gag, gp160, or unanchored gp140 were administered three times to rhesus macaques, followed by a mucosal challenge with SIVsmE660 (73). Macaques immunized with SIV-VEE virus replicon particles produced neutralizing antibodies and had reduced peak viral loads (1 log) postchallenge. In correlation with the decreased viral loads was an increase in the peripheral CD4⁺ T-cell counts in immunized macaques, observed at the viral set point. While not measured in this study, immune responses at mucosal sites, namely the preservation of CD4⁺ T cells, will be interesting to analyze. Overall, however, this platform has progressed very slowly. To date, there is a limited amount of clinical data, and studies to support the efficiency of this platform are remain important.

	VESICULAR STOMATITIS VIRUS

 
A member of the Rhabdoviridae family, vesicular stomatitis virus (VSV) is a negative-stranded RNA virus that primarily infects livestock. In most of the world, infection of humans is rare and asymptomatic but can cause flu-like symptoms. VSV is an attractive platform for clinical use, as it can be delivered at mucosal sites, carry multiple transgenes, is easily manufactured, and has low seroprevalence in humans (reviewed in references 88 and 121). One potential drawback to VSV vaccine vectors is the development of antibodies against VSV surface glycoproteins in immunized hosts after one immunization. Therefore, effective boosting may require substituting a glycoprotein from a different VSV serotype. In nonhuman primates, immunization with rVSV vectors encoding HIV Env and SIV Gag protected against disease after challenges with SHIV 89.6p (41, 43). Furthermore, the direct comparison of intramuscular versus intranasal administrations of equal doses of HIV Env and SIV Gag (5 x 10⁶ PFU each) to rhesus macaques demonstrated that intranasal vaccination elicited higher cellular immune responses, as measured by IFN- ELISPOT assays, Cr release assays, and tetramer analysis (43). Interestingly, there was no difference in the humoral immune responses elicited by either vaccination strategy at systemic or mucosal sites (43). The intranasal delivery of VSV vectors does not seem to cause neurovirulence in nonhuman primates (71). It will be interesting to see if the mucosal administration of this promising platform can elicit cellular immune responses at mucosal sites.

	POXVIRUS VECTORS

 
Poxviruses are large DNA viruses that evolved as vectors based on experience with the smallpox vaccine (reviewed in reference 93). As vaccine vectors, they are able to carry multiple and large genes (25 kb) (97) that are stably expressed. These vectors are also capable of inducing immune responses similar to those observed with the pathogen. Several studies have shown that vaccinia virus vectors can be pathogenic in immunocompromised people, thereby refocusing efforts on developing a more-attenuated, modified vaccinia virus Ankara (MVA) as a vector system (105). MVA has been shown to be safe for mice, monkeys, and humans, as it cannot complete an entire life cycle in humans or primates (41) while still being capable of inducing strong cellular and humoral immune responses in mice and primates. Numerous poxvirus-based vaccines have been tested in rhesus macaques after they were challenged with SIV, SHIV, and HIV-2 isolates (2, 7, 19, 47, 57, 59-62, 114) and have produced various degrees of cell-mediated immune responses and protection against viral challenge.

In a study conducted with rhesus macaques, SHIV DNA plasmid-producing noninfectious viral particles were delivered intranasally, with a rMVA SIV Gag-Pol and HIV Env boost. Coimmunization with an interleukin-2 (IL-2)/Ig plasmid elicited both mucosal and systemic humoral and cellular anti-SHIV immune responses (22). Intranasal immunization with other platforms had previously been reported to be the only route of mucosal vaccination that results in disseminated humoral and cellular immune responses (82, 83) and increases the level of vaginal responses compared to that for rectal immunization (20, 83). Indeed, as nasal immunization also induced greater systemic antibody responses and thus generated both systemic and mucosal immunity, the intranasal route may precede other mucosal routes (83). Mucosal SHIV-specific responses were detected in the IL-2/Ig group, as measured by the levels of IgA in rectal secretions and tetramer-positive T cells in rectal biopsies. Upon rectal challenge with SHIV 89.6p, all of the animals became infected, but the group coimmunized with IL-2/Ig was protected from CD4⁺ T-cell loss and disease progression to AIDS. Interestingly, a different study showed that tetramer-positive CD8⁺ T-cell responses were induced at mucosal sites by live attenuated poxvirus NYVAC encoding SIV Gag, Pol, and Env, whether it was administered by the intranasal, intramuscular, or intrarectal route (140).

A recombinant canarypox vector (ALVAC 1452) has been tested in multiple clinical trials with different doses, formulations, and prime-boost regimens by the HVTN and others. The results of a phase II study of ALVAC and VaxGen's gp120 protein (HVTN 203), demonstrated by IFN- ELISPOT assays, were observed in less than 36% of the vaccinees; therefore, the planned phase III trial (HVTN 501) was cancelled. Results from another phase II trial, outside of the United States, which tested ALVAC with or without a rgp120 booster (HVTN 026), did not show a difference in cellular immunity between the vaccinated and placebo groups, as measured by IFN- ELISPOT assays, lymphocyte proliferation assays, or Cr release assays (35). Increasing the dose of the ALVAC vector (HVTN 039) or the addition of lipopeptides (HVTN 042) did not enhance the number of responders, 5 of 52 and 3 of 77, respectively (37). A phase III clinical trial in Thailand involving the same ALVAC vector expressing subtypes E and B Env (gp41), Gag, protease plus gp160, or Chiron gp120B/E boosting is currently under way (76). In this study, CTL responses have been observed in less than 30% of the recipients, and antibodies, but not broadly neutralizing antibodies, have been observed. This finding may have been expected, as the preceding phase I/II study results showed no statistical difference between cellular responses in the ALVAC prime plus gp120 or gp160 protein boost group and the placebo group (149). Improving the immunogenicity of these experienced vectors is a major focus of the poxvirus field.

	HERPESVIRUS

 
Unlike the viral vectors described above, herpes simplex virus type 1 (HSV-1) is able to persist for essentially the lifetime of the infected host. Active HSV-1 infection occurs at mucosal surfaces, including the mouth and genitals, and the virus remains latent in the nervous system, where it can be periodically reactivated. In addition, both cellular and humoral immune responses are generated in response to HSV-1 infection. These features of HSV-1 highlight its potential utility as a vaccine vector in regard to long-term antigenic expression, immunogenicity, and tropism for mucosal surfaces. Both replication-competent and replication-defective recombinant HSV-1 vectors have been studied as vaccines for HIV-1 in nonhuman primate models. In a study by Murphy et al. (107), the replication capability of HSV-1 did not elicit statistically different results, as measured by antibody levels, CTL activity, or protection against challenge. In fact, two rhesus macaques were protected against a SIVmac239 rectal challenge, one immunized with a replication-competent, and the other a replication-deficient, HSV-1 encoding SIV Env and Nef. However, the replication-competent group had peak viral loads 1.2 logs lower than those of the infected control animals. Clearly, the protection against mucosal challenge and the tropism of HSV-1 for mucosal surfaces warrant further examination of immune responses elicited by the mucosal delivery using this platform.

	BACTERIAL VECTORS

 
The use of recombinant bacteria as vectors for gene delivery has been extensively reviewed elsewhere (94). Here we will touch on two particular platforms that have been used for HIV-1 vaccines in nonhuman primate models, Listeria monocytogenes and Salmonella.

Listeria monocytogenes. A recombinant strain of Listeria monocytogenes has many promising features as a vaccine vector, including a natural tropism for mucosal tissues, an ability to infect APCs, a high level of CD8⁺ T-cell induction, a low prevalence of preexposure in humans, and ease of manipulation and production in culture (reviewed in reference 90). Two independent studies performed with rhesus macaques illustrated the induction of mucosal cellular immune responses with a DNA prime and a Listeria boost, both encoding SIV Gag and Env (26, 109). In a study performed by Boyer et al. (26), the macaques that received the DNA prime-Listeria boost had better protection against an intrarectal challenge of SIV239, measured by control of viral loads for a longer period of time. Neeson et al. (109) further examined the induction of a mucosal SIV Gag-specific cellular immune response by the DNA prime-Listeria boost by demonstrating that Gag-specific CD8⁺ T cells in the peripheral blood coexpress the gut-homing marker B7 in the peripheral blood and home to gut mucosal tissues.

Salmonella. Like Listeria monocytogenes, the natural route of infection by Salmonella species is oral, and therefore, use of the recombinant bacteria to induce mucosal immunity is promising. Recombinant Salmonella vectors have been engineered to utilize the bacterial type III secretion system in order to more efficiently present antigenic peptides via MHC-I and, subsequently, prime CD8⁺ CTLs (127). Priming the immune response with an intragastric administration of the modified Salmonella vector expressing HIV-1 Gag in combination with a MVA Gag booster elicited mucosal immune responses in rhesus macaques (44). This combination, in particular, elicited SIV Gag-specific CD8⁺ T cells that expressed 4β7 and homed to the colonic mucosa. While such responses were unable to confer protection against an intrarectal challenge with SIV239, the elicitation of mucosal cellular responses (albeit minimal) is noteworthy. Improving the immune potency of this platform is important.

	NUCLEIC ACIDS

 
While RNA vaccines have been important in the area of immune therapy for cancer, work in the particular area of an HIV-1 vaccine has been limited. Accordingly, we focus here on the DNA vaccine approach.

DNA has been studied as a vaccine platform for over 15 years. Studies with mice immunized with plasmid antigens yielded promising levels of immunity that, unfortunately, have not been well translated in initial clinical studies. Currently, there is a significant amount of work in the field devoted to improving this platform, as it is appealing on many levels, including large-scale vaccine production, safety, repeat administration (no preexisting or vaccine-induced vector serology), and storage, as a cold chain is not required. DNA vaccines for HIV-1 have been reviewed elsewhere (66). Here we will focus primarily on the optimization of future DNA vaccines.

Genetic level. The optimization of plasmids has yielded a substantial increase in the immunogenicity of plasmid-encoded antigens via enhanced levels of gene expression on a per-cell basis. For example, codon optimization improves expression by adapting the codon usage to the bias of the target species, thereby enhancing transcription through more-abundant tRNA pools (8, 39, 159, 172). Additional modifications, including eliminating mRNA instability and mRNA inhibitory elements, may also be made to the encoded gene sequence to increase the stability of the mRNA (133). The inclusion of a constitutive transport element can enhance RNA stability and the export of RNA from the nucleus. The addition of IgE leader sequences also facilitates expression by improving the loading of mRNA onto the ribosome (159, 164). By removing these negative sequences, avoiding splice sites, removing double-stranded pairing/secondary-structure formation, and including the above sequences, mRNA structures that are more stable and more efficiently transported to the ribosome can be created. As a result, the mRNA is translated more effectively and gene expression is enhanced on a per-cell basis, which can improve immunogenicity.

Molecular adjuvants. As a vaccine strategy, the use of DNA has been suboptimal in producing immunogenicity. In addition to genetic modifications enhancing the expression of the antigenic plasmid, there is a plethora of work on improving the potency of DNA vaccines. One promising technique involves the incorporation of plasmids carrying immunomodulatory genes. Molecular adjuvants reported to induce mucosal immunity include the chemokines CCL19, CCL21, and macrophage inflammatory protein 1 and the cytokines granulocyte-macrophage colony-stimulating factor, IL-2, IL-12, and IL-18 (152). Two recent nonhuman primate studies conducted by Wyeth included codelivery of the plasmid-encoded cytokines IL-12 (129) and/or IL-15 (33) with an SIV Gag DNA construct. The IL-12 plasmid was the best adjuvant, substantially enhancing both cellular (ELISPOT) and humoral (enzyme-linked immunosorbent assay) immunity against HIV-1 Gag. Coimmunization with both IL-12 and IL-15 enhanced humoral, but not cellular, responses, and this version of the IL-15 plasmid did not statistically enhance the responses observed with the antigen alone. Upon challenge with the pathogenic SHIV 89.6p, macaques coimmunized with IL-12 had lower viral loads and a trend toward the preservation of peripheral blood CD4⁺ T cells, although the groups were too small for statistical significance. Similarly, coimmunization with the IL-12 plasmid augmented cellular immune responses, as measured by IFN- and granzyme B levels in cynomologus macaques (24). In a separate study, cynomologus macaques coimmunized with plasmid-encoded SIV Gag and a highly optimized macaque IL-15 had significantly lower viral loads than the antigen-only group following challenge with SHIV 89.6p, even though coimmunization with IL-15 did not significantly enhance the IFN- response compared to that of the antigen alone (25). This study, as well as others, highlights the necessity of supplementing the ELISPOT assay with multiparameter immune response analysis, as well as other assays of T-cell function. Another adjuvant, IL-2, has shown encouraging potential. Coimmunization of rhesus macaques with plasmid-encoded Gag and Env and an IL-2/Ig fusion construct resulted in a potent CTL response and subsequently reduced viral loads, maintained stable CD4⁺ T-cell counts, and prevented disease progression to AIDS (13). Overall, these primate data are exciting. The clinical studies of such approaches are anxiously being followed. In addition to cytokines being codelivered with an antigenic plasmid, other immunomodulatory genes such as costimulatory molecules, including CD40L and glucocorticoid-induced tumor necrosis factor receptor, are being tested in macaques (141, 142), and granulocyte-macrophage colony-stimulating factor (14), B7.1, LFA-3, and ICAM-1 (165) are being tested in mice. Whether there are mucosal benefits to DNA vaccination remains to be seen.

Improvements in delivery. Strategies to improve the dosing, delivery, longevity, and transfection efficiency of DNA vaccines are receiving a great deal of attention. One method of facilitating delivery of plasmid DNA is through formulations that include incorporation of liposomes or polymers to extend plasmid longevity following injection and thereby enhance uptake over time and, subsequently, expression (5, 32, 53, 111). Improved delivery methods include gene gun (49, 145, 153), BioJet (3, 56, 153), laser (168), ultrasound (146), and electroporation (63, 151, 160) approaches to enhance physical delivery. The gene gun is advantageous in that it requires very low amounts of DNA. This method of plasmid delivery has an apparent ability to drive antibody responses in both nonhuman primates and humans, although improving CTLs is an important goal of this technology. Another method, the BioJet, is a needle-free jet injection device that may reduce the variability of responses in primates and humans (3, 56, 153). The use of laser, ultrasound, and electroporation are all based on the permeabilization of cell membranes at the injection site through light, sound, and electric current, respectively.

Various types of in vivo electroporation have also recently been employed to increase the transfection efficiency of plasmid DNA. This technique involves applying a specific electric current to the target, either the muscle or skin, based upon the type of immunization delivered, intramuscular or intradermal. Enhanced delivery is observed and is likely due to the formation of transient pores within the cell membrane, allowing for more cellular uptake of plasmid DNA. Concurrently, an inflammatory response is induced and likely enhances the observed immune response as well (10). In a comparative study of intramuscularly delivered plasmid DNA (IM) versus electroporation, electroporated mice were given 2- to 10-fold less DNA than the IM group. As shown in Fig. 2, the electroporated mice immunized with 25 µg of DNA had levels of IFN- secretion twofold higher than the IM group immunized with 50 µg of DNA without electroporation (approximately 550 versus 250 spot forming cells [SFC], respectively). Furthermore, the electroporated mice immunized with only 5 µg of DNA had levels of IFN- secretion similar to those of mice immunized IM with 50 µg and elicited significantly higher anti-Gag antibody levels than the IM group without electroporation (Fig. 2). In rhesus macaques immunized with plasmid DNA versus electroporation with one-fifth of the DNA, the cellular immune responses of the electroporated group were enhanced by 10- to 40-fold, with antibody titers 2.5 logs greater than those immunized with DNA alone (96). The results of this study put plasmid DNA vaccines for HIV-1 on the potency map. The benefits observed in the primate model include enhanced plasmid delivery, resulting in increased gene expression, and enhanced immunogenicity (64, 113). The combination of electroporation with novel formulations is exciting to consider. Our laboratory has shown a significant enhancement of T-cell proliferation in rhesus macaques that were given an intramuscular DNA immunization with electroporation versus an intramuscular immunization alone (64a). However, there are obvious concerns with this technology versus the standard needle and syringe delivery method, including increased pain, more-complex technology to establish, and requirement of a machine and training for the clinical staff who administer the vaccine. The overall goal will be to decrease the required dose and voltage and, subsequently, to increase the tolerability without sacrificing effectiveness. The inclusion of cytokines (Fig. 3) appears to further lower the dose while simultaneously enhancing the potency of the vaccine and may allow for a decrease in the number of injections required to achieve useful clinical potency.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Electroporation enhances both cellular (A) and humoral (B) immune responses, using less amounts of DNA than intramuscular immunizations alone. BALB/c mice were immunized in the muscle 2 weeks apart with or without electroporation. The group given the intramuscular injection alone (IM) received 50 µg of plasmid DNA, and the electroporated group (IM/EP) received 5 µg or 25 µg per immunization. The mice were sacrificed 1 week following the final immunization. (A) ELISPOT assay with the splenocytes of mice immunized with empty plasmid (pVAX) or pHIV-1 Gag DNA (pGAG). HIV-1 Gag-specific IFN-g secretion is shown as SFC per million. Splenocytes were stimulated with four overlapping peptide pools spanning HIV-1 Gag. (B) Antibodies against p24 HIV-1 Gag, as measured by enzyme-linked immunosorbent assay. Sera were collected from immunized mice at the time of sacrifice. O. D. at 450, optical density at 450 nm.

View larger version (42K): [in this window] [in a new window]   FIG. 3. Electroporation increases CD4 and CD8 T-cell proliferation. Rhesus macaques were immunized three times with 1.0 mg each of plasmid-encoded consensus HIV Gag and rhesus IL-12 by intramuscular injection (IM) immunization alone (n = 2), or electroporation (EP; n = 3). Peripheral blood mononuclear cells were isolated from samples taken 2 weeks after the third immunization, and the proliferative capacities of HIV Gag-specific CD4 and CD8 T cells were determined by carboxyfluoroscein succinimidyl ester (CFSE) assay. Representative dot plots for two animals from each group are shown. Percentages represent Gag-specific proliferation with background proliferation (medium control) subtracted..

	DNA/VECTOR PRIME-PROTEIN BOOST

 
The administration of a DNA prime and a subsequent protein boost effectively induces both cellular and humoral immunity. In rhesus macaques, a polyvalent DNA encoding Env from multiple subtypes and a Gag gene prime plus a homologous gp120 protein boost elicited modest cellular (around 500 SFC) and robust humoral (10⁶ SFC) responses (116). Upon rectal challenge with SHIV-Ba-L, the majority of immunized macaques were protected from infection, while the rest had lower viral loads than the unvaccinated group. A similar human ALVAC-HIV-1 recombinant vaccine expressing Gag, Pol, and gp120 or a Gag, Pol, and gp160 prime plus a homologous Env protein boost resulted in decreased viral load levels in the blood and in mucosal sites while simultaneously protecting macaques from peripheral CD4⁺ T-cell loss after challenge with SHIV_KU2 (115). This approach is being studied in a phase I clinical trial by S. Lu (95) and colleagues, using five different gp120 isolates and one Gag DNA prime plus a boost with heterologous gp120 protein. Interestingly, anti-gp120 antibody titers comparable to those of patients with chronic HIV-1 infection have been reported. In addition, anti-Env cellular responses were induced by this combination.

	DNA PRIME-rAd BOOST

 
rAd5 is also being used as a boost for a unique DNA prime in large-scale clinical trials being conducted by the NIH and VRC. In a phase I trial, DNA and Ad5 were administered separately to 86 recipients at increasing doses. The vast majority of recipients had an anti-HIV-1 T-cell response, the first high responses in published reports (30, 52). Four DNA constructs, clade B Gag-Pol-Nef fusion protein, and clades A, B, C Env glycoproteins (31, 79) elicited an antibody and long-lasting Env-specific CD4⁺ and CD8⁺ T cells. Comparatively, a higher percentage of responders with a greater magnitude of immune responses was observed in recipients of rAd5 (clade B Gag-Pol fusion protein and clades A, B, C Env glycoproteins). Although impressive levels of cellular and humoral immunity were observed in the majority of subjects, preexisting immunity to Ad5 lowered T-cell responses approximately threefold compared to those of seronegative subjects. Based on the safety of, and encouraging results from, this study, a phase II trial has been initiated (124). Recent studies have shown a dramatic increase in HIV-1-specific immune responses with the combination of these two platforms. The DNA prime with a rAd5 boost resulted in a greater-than-1,000-fold increase in antibody and a greater-than-5-fold increase in T-cell responses against Env (124). Data from macaques immunized with this strategy and subsequently challenged with SIV show long-term preservation of the total number and function of central memory CD4⁺ T cells in the peripheral blood as a result of vaccine-generated protection against high levels of virus replication (86, 98). Efficacy trials of this approach are being planned through a HVTN/VRC collaboration.

	DNA PRIME-rAAV BOOST

 
In the previously mentioned rAAV nonhuman primate study by Johnson et al. (72), a DNA prime-rAAV boost protocol elicited potent responses compared to those of other regimens using rAAV vectors alone. The study utilized either a DNA or rAAV prime with a mixture of three plasmids encoding the same SIV subgenomic fragments of (i) Gag, protease, and reverse transcriptase, (ii) reverse transciptase and integrase, and (iii) Rev and Env. Both groups received the same rAAV Rev-Env boost. Despite the weakly immunogenic DNA prime, the monkeys with the lowest viral loads and therefore the best control of SIV replication postchallenge were in the DNA prime and rAAV boost group.

	DNA PRIME-VSV BOOST

 
While preexisting immunity to VSV is not an obstacle for humans, the ability to boost with the same rVSV strain is limited due to antivector immune responses. Therefore, the use of VSV in a heterologous prime-boost regimen with plasmid DNA is an attractive strategy for eliciting more-potent immune responses. In A01-negative rhesus macaques, intramuscular immunization with 5 mg of each DNA plasmid encoding SIV Gag and the cytokine adjuvant IL-12, followed by intranasal delivery of 5 x 10⁶ PFU of each rVSV encoding HIV Env (gp120) and a SIV Gag booster elicited both cellular and humoral immune responses greater than either platform alone (42). In particular, following a high-dose (300 monkey infectious doses) intravenous SHIV 89.6p challenge, this prime-boost regimen resulted in a 2.4-log reduction in viral loads compared to that for the DNA prime (1.5 log) or rVSV boost (1.1 log) given alone (42). In addition to there being no clinical disease observed postchallenge (42), there was enhanced preservation of CD4⁺ T cells in the peripheral blood of these animals.

	DNA PRIME-MVA BOOST

 
One of the most-studied prime-boost regimens is a DNA prime and a poxvirus boost (123, 132). Interestingly, the T-cell responses generated with this heterologous strategy produce immune responses 10 times higher than either platform given separately (106). In fact, an important clinical trial by Robinson and colleagues is under way through the HVTN, utilizing a multivalent DNA (Gag, Pol, Env, Tat, Rev, Vpu) prime followed by a MVA boost. Initial results for humans show exceptional safety and tolerability but undetectable HIV-1-specific cellular (responders = 0/28, as determined by IFN- ELISPOT assay, and 0/24 by Cr release assay [37]) or humoral responses after a DNA prime (106). However, based upon data for rhesus macaques, the animals with minimal responses detected after a DNA prime were able to generate potent immune responses post-MVA boost (138), suggesting that the same may be true for humans. Preliminary reports suggest that the poxviral component increases the levels of immunity observed in these studies. In addition, data from another rhesus macaque study using Gag, Pol, and Env DNA prime plus MVA boost resulted in the control of viremia after a mucosal challenge with SHIV 89.6p (6).

	DNA PRIME-HSV-1 BOOST

 
A recent study with rhesus macaques compared the immune responses elicited by immunization with recombinant DNA and HSV-1-encoding SIV genes in a heterologous DNA prime-HSV-1 boost strategy to those from homologous HSV-1 immunizations (77). Despite the robust cellular and modest humoral immune responses observed in the immunized macaques, all became infected upon an intravenous challenge with SIV239. Interestingly, the macaques immunized with the DNA prime-HSV-1 boost regimen had greater levels of T-cell responses than those of macaques immunized with HSV-1 alone. The observed levels of IFN- secretion for the prime-boost group were greater than or equal to those observed for unvaccinated macaques infected with wild-type SIV. In fact, while the DNA prime-HSV-1 boost regimen also produced a higher percentage of tetramer-postive IFN--secreting cells, the group that received the homologous HSV-1 immunizations had more 4β7-positive cells, a marker of T cells homing to the mucosa. We look forward to future studies addressing the ability of HSV-1 to induce mucosal immune responses.

	CONCLUSIONS

 
It is a tumultuous time for HIV vaccine development. There are few promising leads in the vaccine toolbox for antibody-based approaches. The STEP study has thrown cold water on the cadre of CTL-based platforms. These approaches (summarized in Table 2) provide the broadest array of T-cell-driving vectors in the history of vaccinology. In the near future, additional prime-boost studies and the use of adjuvanted DNA, designer Ad vectors, and DNA electroporation strategies will be expanded in the clinic. The world's first glimpse of the lack of efficacy of a pure T-cell-based approach supports new research goals for the CTL-based HIV vaccine field. (i) More-stringent primate models for determining vaccine efficacy in decision studies should be encouraged. (ii) We should demand greater CTL responses in humans, greater than an average of 300 to 400 10⁶ peripheral blood mononuclear cells, from next-generation is approaches. (iii) The induction of T-helper responses T-cell desirable and likely important, but we need to learn a great deal more about their contributions to immune control. (iv) The importance of polyfunctional immune responses, in addition to the proliferative capacity of vaccine-elicited T-cell responses, should be studied, and benchmarked as they relate to vaccine outcome in macaques and immune control in humans. (v) Novel and clean hypotheses regarding vectors entering clinical trials should be encouraged, and immune evaluations should be designed on the basis of these hypotheses and serve as benchmarks for progress in the clinic. (vi) Most importantly, rather than redividing the limited DAIDS funding pie, thus depriving important programs of needed resources, Congress should support the NIH by increasing funding for basic HIV vaccine research and pilot programs for early clinical testing. New ideas and concepts cannot be advanced effectively based on the current levels of funding for R01's, P01's, and developmental resources.

	ACKNOWLEDGMENTS

 
K.A.S. acknowledges the support of NIH grant 1-T32-A107632. D.B.W. acknowledges support from the NIH, including HIVRAD funding.

	FOOTNOTES

 
* Corresponding author. Mailing address: 505 Stellar-Chance Laboratories, 422 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104-6100. Phone: (215) 662-2352. Fax: (215) 964-5885. E-mail: dbweiner{at}mail.med.upenn.edu

Published ahead of print on 7 November 2007.

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