Effect of Preexisting Immunity on Oncolytic Adenovirus Vector INGN 007 Antitumor Efficacy in Immunocompetent and Immunosuppressed Syrian Hamsters

Immune responses against adenovirus (Ad) vectors pose a possible concern for the outcome of treatment efficacy. To address the role of preexisting immunity in oncolytic Ad vector antitumor efficacy following intratumoral injection of vector as well as tumor-to-tissue spread of the vector, we employed the Syrian hamster model. These animals are immunocompetent, and their tumors and tissues are permissive for replication of Ad type 5 (Ad5). We used the adenovirus death protein-overexpressing Ad5-based vector INGN 007. Subcutaneous tumors were established in groups of hamsters that were or were not immunized with Ad5. Half of the hamsters in these groups were immunosuppressed with cyclophosphamide. For all groups, tumors injected with INGN 007 grew significantly more slowly than those injected with buffer. Under immunocompetent conditions, there was no significant effect of preexisting immunity on vector antitumor efficacy. Soon after the tumors in naïve animals were injected with vector, the hamsters developed neutralizing antibody (NAb) and the difference in NAb titers between the naïve and immunized groups diminished. Under immunosuppressed conditions, preexisting NAb did significantly reduce vector efficacy. Thus, NAb do reduce vector efficacy to some extent, but immunosuppression is required to observe the effect. Regarding vector toxicity, there was spillover of vector from the tumor to the liver and lungs in naïve immunocompetent hamsters, and this was nearly eliminated in the immunized hamsters. Thus, preexisting immunity to Ad5 does not affect INGN 007 antitumor efficacy following intratumoral injection, but immunity prevents vector spillover from the tumor to the liver and lungs.Oncolytic (replication-competent) viral vectors are being investigated as a treatment for cancer (2, 19, 25, 27). Recently, an oncolytic adenovirus serotype 5 (Ad5)-based vector was approved for cancer therapy in humans for the first time (14, 42). Oncolytic vectors based on Ad, reovirus, herpes simplex virus type 1 (HSV-1), poxvirus, poliovirus, Newcastle disease virus, measles virus, and vesicular stomatitis virus (VSV) are being studied extensively in both preclinical and clinical settings (16, 20, 24). Oncolytic Ad vectors are popular due to the Ad safety profile and ease of manipulation and handling (6, 13, 18, 23).Oncolytic Ad vectors infect and kill cancer cells as a result of the normal Ad life cycle by replicating in cells and releasing progeny viruses. These vectors rely on replication and spread through the tumor to achieve efficacy. A majority of the human population is seropositive for Ad5, which is acquired as a childhood infection (4, 15, 39). Elimination of the vector by preexisting immunity to Ad or vector elimination by the adaptive immune response generated after administration of the vector poses a possible concern with respect to achieving significant antitumor efficacy. A key question is whether the oncolytic Ad vector can efficiently eliminate tumor cells faster than its own clearance by the immune system. Several studies show that suppressing the immune system enhances the efficacy of oncolytic vectors (10, 12, 31).Alternatively, studies show that activation of the adaptive immune system by the vector might increase tumor cell killing, thereby increasing vector antitumor efficacy (11, 21, 27, 34). Studies with oncolytic HSV and VSV show that these vectors induce long-term antitumor immunity (11, 21, 27, 34). Therefore, apart from direct cell lysis, oncolytic vectors may be able to achieve antitumor efficacy by activating the antitumor immune response. Therefore, induced or preexisting immunity to the vector can be either a hurdle or beneficial for vector efficacy.Most efforts to address the effect of preexisting immunity were performed by gene transfer studies with replication-defective Ad vectors (28, 41). These studies showed that preexisting immunity significantly reduces gene transfer and expression in the target organ. In contrast, other studies showed that preexisting immunity does not prevent gene transfer (26) and does not affect vector antitumor efficacy (1). Little work has been done to address the role of induced or preexisting immunity on the efficacy and toxicity of oncolytic Ad vectors (3, 39). Studies with these vectors have been difficult because of a lack of immunocompetent and permissive animal models. Ad replication is generally species specific, and human Ads replicate poorly in cells from most nonhuman species. Consequently, Ad vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model cannot adequately address the effect of the host immune system on the vector-infected tumor or the toxicity of the vector in normal tissues.We recently developed a novel Syrian hamster model for the study of oncolytic Ad5-based vectors (30). These animals are both replication permissive for Ad5 and immunocompetent. In the present study, we modeled the effect of preexisting immunity to Ad5 on the efficacy of an oncolytic Ad vector, INGN 007, and the spillover of the vector from the site of injection to the liver and lungs.     

Effect of Preexisting Immunity on an Adenovirus Vaccine Vector: In Vitro Neutralization Assays Fail To Predict Inhibition by Antiviral Antibody In Vivo

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8⁺ T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector''s capacity to transduce cells and to stimulate a transgene product-specific CD8⁺ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.Adenovirus (Ad) vectors are effective at inducing potent CD8⁺ T-cell responses to immunogens. In animal models, Ad vectors encoding antigens of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV), used in combination with plasmid-based DNA vectors, generate CD8⁺ T-cell responses that attenuate infection by SIV (9) and by HIV-SIV chimeras (16). In humans, Ad vectors derived from human serotype 5 (AdHu5) are immunogenic and are well tolerated at immunogenic doses; however, in a recent clinical trial, an AdHu5-based HIV-1 vaccine failed to prevent (and may have facilitated) infection (1a). It is not clear whether CD8⁺ T-cell responses will be sufficient to prevent or control HIV infection and disease. However, it seems likely that the induction of effective immune responses against HIV will require multiple doses of antigen, with a priming dose followed by one or more booster immunizations. Prime-boost regimens based on the sequential use of DNA and AdHu5 vectors are being tested clinically, and regimens involving the sequential administration of serologically distinct Ad vectors are being explored in preclinical animal models (1, 5, 8, 9).One major obstacle to the use of vectors derived from AdHu5 and other common human serotypes is the high prevalence of virus-neutralizing antibodies (VNAs) in humans. Preexisting VNAs to the vaccine carrier prevent the vector from transducing target cells, which reduces the amount of vaccine antigen that can be produced and dampens the resultant adaptive immune responses (2, 3, 12). Approximately 40 to 45% of the U.S. population has VNAs to AdHu5, and seroprevalence rates are even higher in Asia and Africa (6, 24).We developed vectors derived from chimpanzee Ads to which humans lack preexisting immunity. When tested in a rodent model, one such vector, AdC68, induces potent transgene product-specific CD8⁺ T-cell responses that can be increased by booster immunizations with serologically distinct Ad vectors (3, 19, 23). However, because the use of multiple serotypes in a prime-boost regimen may prove cumbersome in clinical applications, we have attempted to modify the major neutralizing binding sites within the AdC68 capsid. It has been suggested that the binding sites for Ad-neutralizing antibodies preside primarily within the major capsid protein hexon (4, 10, 14, 15, 17). We defined a single hexon surface loop as the major neutralization site on AdC68 and showed that a mutant vector, AdCDQ, which incorporates a 3-amino-acid mutation within this loop, resists in vitro neutralization by polyclonal antisera obtained from animals immunized against AdC68 (10). Because it is serologically distinct from its parent vector, we expected that AdCDQ could be used in combination with AdC68 in an effective prime-boost regimen.In the present study, we tested whether the AdCDQ vector induces a transgene product-specific CD8⁺ T-cell response in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ vector in vitro nevertheless impair the vector''s capacity to transduce cells and to stimulate a transgene product-specific CD8⁺ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on Ad vectors in vivo.     

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV^CAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV^CAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV^CAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV^CARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV^CAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV^CAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV^CAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV^CAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV^CAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV^CAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV^CAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV^CAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.Adenoviruses (Ads) are extensively used today as gene transfer vectors for in vitro, ex vivo, and in vivo gene transfer protocols (reviewed in reference 65). Cell entry of human Ad type 5 (Ad5), the serotype most widely used as a gene vector, occurs most efficiently by the receptor-mediated endocytosis pathway (reviewed in references 64 and 65), via the coxsackievirus B-adenovirus receptor (CAR) (3, 77) and αvβ3/αvβ5 integrins (84, 85), although alternative receptors have been described (11, 12, 14, 27). Cell surface expression of CAR differs with different cell types, and this represents one of the major determinants of the efficiency of Ad5-mediated transduction (43). The ubiquitous nature of CAR is responsible for transduction of nontarget tissues by Ad vectors. Paradoxically, many target cells such as dermal fibroblasts, synoviocytes, mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and dendritic cells (DCs), express no or very low levels of CAR at their surface and are relatively resistant to Ad transduction (14, 15, 19). Much work has been done with different strategies to promote the entry of Ad5 into CAR-defective cells. These strategies include (i) the genetic modification of Ad capsid proteins to carry cell ligands (2, 15, 20, 28, 49, 50), (ii) pseudotyping Ad5 vectors with fibers from other serotypes (13, 57, 74, 86), (iii) using bispecific adapters or peptides (25, 40), (iv) chemical modification of Ad (9, 42), and (v) tethering on nanoparticles (7). The limitations to these strategies are that modifications of the Ad capsid are susceptible to negatively affecting the virus growth or viability, due to an alteration of virion assembly, stability, the viral uncoating process, and/or intracellular trafficking (13, 51).Other viruses which are gaining popularity as gene transfer vectors are the baculoviruses (BVs). Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) is an insect virus with a large double-stranded DNA genome packaged in a membrane-enveloped, rod-shaped protein capsid (70). Since the 1980s, the BV-insect cell expression system has been highly exploited for the production of recombinant proteins. In the mid-1990s, it was shown that recombinant BVs carrying reporter genes under cytomegalovirus (CMV) or retroviral Rous sarcoma virus promoter efficiently expressed reporter genes in mammalian cells (6, 22, 38, 41, 44, 69), as well as in avian cells (72) and fish cells (45). Since then, BVs have been reported to transduce numerous cells originating from species as various as humans, bovines, and fish (8, 32, 41, 73). As gene transfer vectors, BVs have been found to be rapidly inactivated by human serum complement (23), but exposing decay-accelerating factor (DAF) at the surface of BV by fusion with the baculoviral envelope glycoprotein can overcome this inactivation (33). BVs also have a good biosafety profile due to their incapacity to replicate in mammalian cells (31).Taking advantage of the ability of BVs to transduce a large repertoire of cells of invertebrate and vertebrate origins, including human primary cells, we investigated whether a recombinant AcMNPV could act as a carrier or macroadapter for Ad5 vectors to enter Ad5-refractory cells. To this aim, we pseudotyped AcMNPV virions with the high-affinity receptor for Ad5, the human CAR glycoprotein (BV^CAR), to enable the formation of complexes between vector particles of BV^CAR and Ad5-green fluorescent protein (Ad5GFP) mediated by Ad5 fiber and CAR interaction. We found that transduction of cell lines which were poorly permissive to Ad5, including human cancer cells and primary cells, was significantly improved using this strategy of piggybacking Ad5 vector on BV^CAR. More importantly, the increase in BV^CAR-Ad5-mediated transduction was obtained with a low range of Ad5 inputs, i.e., at multiplicities of infection (MOI) of less than 50 Ad5 vector particles per cell. We also found that the cell transduction enhancement observed with BV^CAR-Ad5 required the direct coupling of Ad5 to BV^CAR via fiber-CAR binding and that the cell attachment of the complex was mediated by the baculoviral envelope glycoprotein gp64. Kinetic analysis of virus-cell binding showed that the presence of BV^CAR in the complex was beneficial to Ad5 vector, not only in terms of tropism but also in terms of number of cell-bound virions and rate of cell attachment. In addition, the endocytic pathway of BV^CAR-Ad5 did not require Ad5 penton base RGD-integrin interaction. When administered in vivo to nude BALB/c mice, BV^CAR-Ad5 complex showed the same biodistribution as that of control Ad5 vector injected alone. In vitro, transduction of CAR-negative cells by BV^CAR-Ad5 was insensitive to coagulation factor X (FX), in contrast to Ad5 vector alone.Our novel strategy of gene delivery using the BV^CAR-Ad5 duo could be advantageously applied to various situations in vitro or ex vivo, e.g., for transducing Ad5-refractory cells when Ad5 capsid modifications cannot be envisaged, when oncolytic Ads need to be delivered to tumors via nonpermissive cell carriers belonging to the immune system, or when the simultaneous delivery of two transgenes by two separate vectors might be beneficial in terms of timing and/or level of cellular expression of the transgene products.     

Adenovirus-Based Vaccines: Comparison of Vectors from Three Species of Adenoviridae

In order to better understand the broad applicability of adenovirus (Ad) as a vector for human vaccine studies, we compared four adenovirus (Ad) vectors from families C (Ad human serotype 5 [HAdV-5; here referred to as AdHu5]), D (HAdV-26; here referred to as AdHu26), and E (simian serotypes SAdV-23 and SAdV-24; here referred to as chimpanzee serotypes 6 and 7 [AdC6 and AdC7, respectively]) of the Adenoviridae. Seroprevalence rates and titers of neutralizing antibodies to the two human-origin Ads were found to be higher than those reported previously, especially in countries of sub-Saharan Africa. Conversely, prevalence rates and titers to AdC6 and AdC7 were markedly lower. Healthy human adults from the United States had readily detectable circulating T cells recognizing Ad viruses, the levels of which in some individuals were unexpectedly high in response to AdHu26. The magnitude of T-cell responses to AdHu5 correlated with those to AdHu26, suggesting T-cell recognition of conserved epitopes. In mice, all of the different Ad vectors induced CD8⁺ T-cell responses that were comparable in their magnitudes and cytokine production profiles. Prime-boost regimens comparing different combinations of Ad vectors failed to indicate that the sequential use of Ad vectors from distinct families resulted in higher immune responses than the use of serologically distinct Ad vectors from the same family. Moreover, the transgene product-specific antibody responses induced by the AdHu26 and AdC vectors were markedly lower than those induced by the AdHu5 vector. AdHu26 vectors and, to a lesser extent, AdC vectors induced more potent Ad-neutralizing antibody responses. These results suggest that the potential of AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human Ads.Due to their ability to induce potent transgene product-specific B- and T-cell responses, replication-defective adenovirus (Ad) vectors are being explored for use as carriers of vaccines for a variety of pathogens, including human immunodeficiency virus type 1 (HIV-1) (7), Plasmodium falciparum (9), and Mycobacterium tuberculosis (20). Initial enthusiasm for the use of Ad vectors based on Ad human serotype 5 (AdHu5) was dampened by the finding that preexisting antibodies to this virus, which are found in ∼40% of humans residing in the United States and up to 90% of humans residing in some African countries (28), can reduce transgene product-specific immune responses (16) by reducing vector uptake (19). Enthusiasm further decreased after the phase IIb STEP trial, in which an AdHu5 vector was tested for induction of protection in cohorts at high risk for HIV-1 infection. The vector failed to show efficacy in reducing acquisition rates or lowering viral loads in individuals who became infected and instead appeared to increase susceptibility to infection in humans with preexisting neutralizing antibodies to the vaccine carrier (4). As a result of these setbacks, the use of Ad vectors based on other less common serotypes of human Ads (1) or Ads isolated from different species, such as chimpanzees (21, 25), bovines (24), and canines (31), to circumvent preexisting neutralizing antibodies is being explored. Of these, vectors based on adenovirus family D (AdHu26) were shown to have a low seroprevalence in some countries (1) and are now viewed as promising carriers for Ad vector-based gene transfer.A number of studies showed that AdHu26 vectors are highly immunogenic in nonhuman primates (NHPs), where they induced potent transgene product-specific CD8⁺ T-cell responses (13) that, when they were combined in a prime-boost regimen with an AdHu5 vector expressing gag of simian immunodeficiency virus (SIV), achieved a sustained reduction in viral loads upon SIV challenge of vaccinated animals (14). Intriguingly, AdHu26 vectors have been shown to induce a CD8⁺ T-cell response in NHPs that is qualitatively superior to that induced by AdHu5 vectors. AdHu26-induced CD8⁺ T cells showed a broader response, recognizing more epitopes within the transgene product, and had a more polyfunctional response, in that vector-induced individual CD8⁺ T cells produced multiple factors rather than predominantly gamma interferon (IFN-γ) only (13). This suggests that AdHu26 may have fundamental differences in immunogenicity from other Ad vectors.To elucidate this further, we developed a molecular clone of AdHu26 and a number of recombinant AdHu26 vectors from which E1 was deleted and used these to test human samples for the prevalence of AdHu26-neutralizing antibodies and responding CD4⁺ and CD8⁺ T cells. In addition, we conducted a series of studies with mice to determine if this species showed an immune response to a transgene product delivered by an AdHu26 vector markedly different from that induced by the same transgene product delivered by other Ad vectors. Our results showed that AdHu26, strictly speaking, is not a rare serotype, especially in African countries, where the seroprevalence rates of antibodies to AdHu26 are high. Similarly, most humans carry AdHu26-reactive T cells, which in some individuals are present at very high frequencies. In mice, AdHu26 induces potent CD8⁺ T-cell responses that are quantitatively and qualitatively similar to those induced by other Ad vectors. AdHu26 and chimpanzee-origin Ad (AdC) vectors stimulated only marginal transgene product-specific B-cell responses in comparison to those stimulated by AdHu5 vectors but induced more potent neutralizing antibodies to their capsid antigens.     

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8⁺ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8⁺ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses.The initial interest in vectors based on adeno-associated viruses (AAV) was for applications in gene therapy. Most of the initial work was with vectors derived from AAV serotype 2 (AAV2), which is one of the six initial isolates. In the first in vivo studies, several groups showed stable expression of the transgene Escherichia coli β-galactosidase following intramuscular (i.m.) injection of AAV2-LacZ without immune responses to the transgene (23, 44). The apparent tolerance of the host to AAV-encoded antigens to a variety of transgene products has been demonstrated in mice and some large animals (1, 35, 39). Several mechanisms have been proposed to explain the lack of T-cell responses following in vivo gene transfer with AAV, including ignorance (inadequate presentation of antigen), anergy, and suppression (1, 5, 18, 37).As applications of AAV vectors for in vivo gene transfer expanded, it became clear that the apparent immune privilege of AAV transgene products was not absolute. A number of examples emerged in which the host mounted vibrant T-cell responses to AAV-encoded transgene products. Several key parameters appeared to influence immunogenicity of the transgene. For example, Sarukhan et al. suggest that the subcellular localization of the protein influences the magnitude of the ensuing T-cell response after AAV gene transfer (37). The dose and route of administration of the AAV vector also contribute significantly to B- and T-cell responses to the transgene (3, 13). Wang et al. showed that inflammation at the site of AAV administration promotes antigen-specific immune responses to the transgene (47). A consistent observation has been that B-cell responses to AAV-encoded transgenes are much more intense and more consistently generated than CD8⁺ T-cell responses (8, 46, 51). A number of investigators have begun to explore AAV vectors as genetic vaccines against a variety of infectious and noninfectious diseases, based on the notion that it can be developed to stimulate transgene immune responses (14, 22, 26, 28, 48-50).The discovery of an expanded family of AAV capsids from human and nonhuman primates has provided an opportunity to evaluate the effects of capsid structure on vector performance. Most of this work has focused on the use of novel AAV serotypes for achieving higher levels of transgene expression for applications in gene therapy (7, 12, 36). Xin et al. recently evaluated, in mice, vectors as vaccines for human immunodeficiency virus type 1 (HIV-1) based on the original AAV isolates AAV1 to AAV6 and two novel AAVs we recently discovered, AAV7 and AAV8 (48). They showed significant capsid-dependent effects on T- and B-cell responses to HIV-1 gp160. We recently confirmed these observations and more thoroughly evaluated the quality of the CD8⁺ T-cell responses (26). AAV vectors of multiple serotypes encoding HIV-1 Gag were injected i.m. into mice, which all showed some level of CD8⁺ T-cell responses based on tetramer staining and peptide-induced gamma interferon (IFN-γ) expression. However, the quality of AAV-induced, Gag-specific T cells was substantially lower than that obtained with adenoviral vectors, based on several criteria. A majority of the tetramer-positive (Tet⁺) T cells were nonresponsive to antigen, and those that did respond to antigen produced low levels of IFN-γ and no interleukin-2 (IL-2). Very few memory T cells were generated, and animals primed with AAV vectors were not responsive to a boost with an adenoviral vector. However, all AAV serotypes studied did generate very high levels of antibodies to the Gag transgene product.A final issue to consider in the use of AAV as a genetic vaccine for HIV-1 is the presence of neutralizing antibodies (NAbs) to the vector due to prior AAV infections. We recently conducted an extensive screening of human populations from several continents and found high prevalence and high titers of NAbs to AAV1 and AAV2 and moderate levels of NAbs to AAV7 and -8 (4). In vivo gene transfer experiments indicate that AAV NAbs will likely impinge on vector efficacy (9, 33, 38).This study describes the creation of a novel AAV from rhesus macaque isolates, called AAVrh32.33, and its characterization as a genetic vaccine for HIV-1. AAVrh32.33 has properties unlike those of any others we have studied. We showed that vectors based on this novel capsid elicit strong CD8⁺ T-cell responses to reporter transgene products that are dependent on CD4⁺ T-cell help and dependent on signaling through CD40L and CD28 (L. E. Mays and J. M. Wilson, submitted for publication). Important to the use of this vector in the clinic is a very low incidence of NAbs to it in human populations. This study describes the development of vectors based on AAVrh32.33 as genetic vaccines.     

Characterization of a Single-Cycle Rabies Virus-Based Vaccine Vector

Recombinant rabies virus (RV)-based vectors have demonstrated their efficacy in generating long-term, antigen-specific immune responses in murine and monkey models. However, replication-competent viral vectors pose significant safety concerns due to vector pathogenicity. RV pathogenicity is largely attributed to its glycoprotein (RV-G), which facilitates the attachment and entry of RV into host cells. We have developed a live, single-cycle RV by deletion of the G gene from an RV vaccine vector expressing HIV-1 Gag (SPBN-ΔG-Gag). Passage of SPBN-ΔG-Gag on cells stably expressing RV-G allowed efficient propagation of the G-deleted RV. The in vivo immunogenicity data comparing single-cycle RV to a replication-competent control (BNSP-Gag) showed lower RV-specific antibodies; however, the overall isotype profiles (IgG2a/IgG1) were similar for the two vaccine vectors. Despite this difference, mice immunized with SPBN-ΔG-Gag and BNSP-Gag mounted similar levels of Gag-specific CD8⁺ T-cell responses as measured by major histocompatibility complex class I Gag-tetramer staining, gamma interferon-enzyme-linked immunospot assay, and cytotoxic T-cell assay. Moreover, these cellular responses were maintained equally at immunization titers as low as 10³ focus-forming units for both RV vaccine vectors. CD8⁺ T-cell responses were significantly enhanced by a boost with a single-cycle RV complemented with a heterologous vesicular stomatitis virus glycoprotein. These findings demonstrate that single-cycle RV is an effective alternative to replication-competent RV vectors for future development of vaccines for HIV-1 and other infectious diseases.The global spread of HIV-1 represents one of the most significant pandemics to afflict humans (22). Despite tremendous efforts to increase HIV awareness in the general population, UNAIDS reports that fewer than one in five people has access to HIV prevention strategies and many are subject to cultural stigmas thwarting such efforts (43). As such, an HIV vaccine is paramount for preventing disease transmission. It is not yet clear precisely what characteristics are critical for an effective HIV vaccine, yet evidence suggests one would need to induce both antibody and CD8⁺ T-cell-mediated immunity (reviewed in reference 25). Live viruses are at the forefront of HIV vaccine development (7) because they are powerful inducers of both of these arms of immunity. We previously demonstrated that replication-competent rabies virus (RV)-based vectors can induce long-lasting antigen-specific immune responses in both murine and monkey models, as well as protect rhesus macaques from an AIDS-like disease (23, 24, 26-29, 42). However, there are safety concerns with the use of any replication-competent virus for widespread immunization. To address this, we sought to develop and evaluate the immunogenicity of a safer alternative: a single-cycle RV expressing HIV-1 Gag as a model antigen.Single-cycle viral vectors are defective in certain viral components that are required for infectious particle assembly (reviewed in reference 12). As such, the virus undergoes one complete cycle of replication in the primary infected cell and produces progeny virions that are unable to spread to a second round of cells. The progeny are noninfectious and provide inert antigen that may or may not be immunogenic (12). In contrast, so-called replication-deficient viruses do not complete that initial round of replication. These two attenuation strategies have been adopted for use with many different viruses including, but not limited to, adenovirus (Ad), vaccinia virus (VV), canarypox virus (CPV), herpes simplex virus (HSV), vesicular stomatitis virus (VSV), and, more recently, RV (4, 6, 9, 18, 21, 33, 35, 36, 38). However, the results regarding the immunogenicity of such vectors are mixed. For example, both the replication-deficient Ad5 vector and modified vaccinia Ankara (MVA) showed reduced humoral and cellular immunogenicity compared to their replication-competent counterparts, but the use of higher titers and multiple immunizations did increase such responses (18, 33, 35). In the case of CPV, the replication-deficient vector provided poor HIV-specific cellular responses, causing the termination of phase II HIV-1 vaccine trials (38). In contrast, single-cycle VSV, a rhabdovirus closely related to RV, has been shown to induce HIV-1 Env-specific CD8⁺ T-cell responses equivalent to full-length VSV when administered intramuscularly (36). However, protection of rhesus macaques against highly pathogenic simian immunodeficiency virus (SIV) challenge by both replication-competent and single-cycle VSV needs to be shown.In the study described here, we generated a single-cycle RV vector expressing HIV-1 Gag (SPBN-ΔG-Gag) by deletion of the entire RV glycoprotein (RV-G) from the RV genome. RV-G was chosen due to its critical role in the attachment and entry of RV into host cells, which makes RV-G one of the most important determinants of viral pathogenicity (10, 11, 37). RV particles lacking G are unable to spread, as evidenced by intracranial infection with a G-deleted RV that remains restricted to the primary infected neurons (13, 44). It must be noted that in the absence of RV-G, virions are still capable of budding though at a 30-fold lower efficiency (32). These virions, however, are incapable of attachment and entry into a secondary host cell. Because of this, SPBN-ΔG-Gag was propagated on a trans-complementing cell line induced to express RV-G (or VSV-RV-G), effectively facilitating virus spread. To evaluate the immunogenicity of the single-cycle vector, we immunized mice and compared the humoral and cellular responses to responses generated by replication-competent RV. Our results indicate that single-cycle RV generates reduced vector-specific antibody responses but similar HIV-1 Gag-specific CD8⁺ T-cell responses. Moreover, these responses can be significantly enhanced by a heterologous boost with a single-cycle RV complemented with a VSV glycoprotein. Taken together, the results presented here show evidence that single-cycle RV is a promising platform for a safe, live viral vaccine for use against HIV-1 and other applications.     

Efficacy of Multivalent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01⁺/B*17⁻ Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01⁺ cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.There is a significant body of evidence suggesting that anti-human immunodeficiency virus type 1 (HIV-1) cellular immunity plays a prominent role in controlling viral infection and progression to disease (15, 32, 33). This stimulated substantial research into vaccines capable of eliciting this type of immunity, and several vaccine candidates (5, 6, 8-13, 22, 29-31, 35) have reached various stages of clinical development. However, the viability of this general vaccine approach was recently undermined by the findings in a phase II trial (called the Step Study) that immunization with a replication-defective adenovirus serotype 5 (Ad5) vaccine expressing HIV-1 clade B Gag, Pol, and Nef was not effective in either reducing acquisition rates and/or lowering set point viral loads in infected subjects (2, 25). In fact, more infections were originally observed in the vaccine group than in the placebo arm (2).The outcomes of the Step Study led to several important questions. Do the results argue against the concept of a HIV-1 vaccine based on the induction of specific T lymphocytes? On the other hand, if cytotoxic T-lymphocyte (CTL) responses are intrinsically valuable for an effective vaccine, what are the shortcomings in the vaccine-induced immunity that contributed to the lack of efficacy in the Step trial? What is the predictive value of preclinical challenge studies for selection of future clinical vaccine candidates? The potential role of CTL responses in an effective vaccine is also challenged by the recently reported phase III study results for the ALVAC vCP1521 prime-AIDSVAX B/E boost vaccine. The efficacy of this vaccine in a low-risk population was recently shown to trend toward prevention of HIV acquisition and not reduction of viral loads (30). Unlike the Step study vaccine, the ALVAC/AIDSVAX vaccine approach utilized a heterologous prime-boost regimen and contained an Env component that may have contributed to the type of outcome observed here. A better understanding of the immune correlates for this vaccine may be possible following further experimental investigations of the samples collected from the phase III study and earlier-stage trials.Despite the proven efficacy of Ad5 vaccination against simian-human immunodeficiency virus 89.6P (SHIV89.6P) challenge, subsequent primate studies provided equivocal results. In a homologous prime-boost regimen, Ad5 vaccine expressing Gag was ineffective against a high-dose simian immunodeficiency virus SIVmac239 challenge (4, 24). The same study compared this regimen with the DNA prime/Ad5 boost regimen that was found to be efficacious in Mamu-A*01⁺ monkeys; the level of protection in the overall study was correlated with the breadth of epitopes recognized and the frequency of induced antigen-specific CTLs. In this study, we examine whether the expansion of antigens to include Pol, Nef, and Env gp140 using the Ad5/Ad5 regimen would improve the outcome against the same high-dose SIV challenge. Of particular interest is the combination of Gag, Pol, and Nef, for which the homologous human vaccine was utilized in the Step study (29).     

Delivery of Human Immunodeficiency Virus Vaccine Vectors to the Intestine Induces Enhanced Mucosal Cellular Immunity

Effective vaccines for human immunodeficiency virus type 1 (HIV-1) will likely need to stimulate protective immunity in the intestinal mucosa, where HIV-1 infection causes severe CD4⁺ T-cell depletion. While replication-competent recombinant adenovirus (rAd) vectors can stimulate adenovirus-specific mucosal immunity after replication, oral delivery of replication-defective rAd vectors encoding specific immunogens has proven challenging. In this study, we have systematically identified barriers to effective gut delivery of rAd vectors and identified sites and strategies to induce potent cellular and humoral immunity. Vector-mediated gene transfer by rAd5 was susceptible to low-pH buffer, gastric and pancreatic proteases, and extracellular mucins. Using ex vivo organ explants, we found that transduction with rAd5 was highest in the ileum and colon among all intestinal segments. Transgene expression was 100-fold higher after direct surgical introduction into the ileum than after oral gavage, with rAd5 showing greater potency than the rAd35 or the rAd41 vector. A single immunization of rAd5 encoding HIV-1 gp140B to the ileum stimulated potent CD8⁺ T-cell responses in the intestinal and systemic compartments, and these responses were further enhanced by intramuscular rAd5 boosting. These studies suggest that induction of primary immune responses by rAd5 gut immunization and subsequent systemic boosting elicits potent antigen-specific gut mucosal responses.Human immunodeficiency virus type 1 (HIV-1) infection is characterized by uncontrolled virus replication and cytopathicity in the intestinal mucosa, the site of major T-cell depletion after primary infection. The gastrointestinal (GI) tract is the predominant site of a pronounced CD4⁺ T-cell loss in the early stages of HIV infection and simian immunodeficiency virus (SIV) infection in the nonhuman primate model (3, 23, 26, 43). It has been suggested that a mucosal vaccine which generates HIV-specific CD8⁺ T cells in the gut could prevent the loss of CD4⁺ cells in gut-associated lymphoid tissue, establishment of infection, or spread of virus (13, 34). Therefore, targeted delivery of vaccines to the GI tract to stimulate mucosal responses has the potential to improve the efficacy of immune protection against HIV-1; however, the site of gene-based transduction and the barriers to vaccine delivery have not been well defined.Adenoviruses (Ads) have been used extensively as vectors for both gene transfer and vaccine development. They offer several advantages as tools for vaccine delivery, such as the ability to transduce both dividing and nondividing cells, relative safety and stability in vivo, ease of production in high titers, and lack of integration (2, 35). These vectors are promising because parenteral administration in both animals and humans has been shown to generate strong and long-lasting humoral and cellular immune responses. The immune responses surpass those achieved with other types of gene vectors and genetic vaccines (5, 38, 46). As a result, recombinant Ad (rAd) vectors have been developed and tested as vaccine vehicles to immunize against a number of pathogens (4, 10, 15, 18, 41).Orally (p.o.) delivered vaccines are attractive in theory because of their ease of administration and potential to deliver antigen to gut-associated lymphoid tissue, permitting induction of immune responses in both mucosal and systemic compartments. At the same time, p.o. delivery of replication-defective rAd vectors has posed a challenge and has met with variable levels of success. Immunization with rAd5 encoding rabies virus antigens, influenza virus antigens, or other antigens has generated some protection against infection in animal models (9, 27, 31, 39, 41), but p.o. immunization has elicited much lower CD8⁺ T-cell responses than systemic delivery (33), and a much higher dose is required to induce immune responses (37). We have recently shown in an HIV vaccine model that rAd41, a human enteric Ad-based vector, induced potent CD8⁺ T-cell responses in both systemic and mucosal compartments when primed p.o. or in the ileum (17). The previous study showed that rAd41 vectors delivered through direct ileal injection elicited mucosal cell immunity, but whether other rAd vectors could stimulate these responses and which factors affected delivery and immunogenicity were unknown. In this report, we have investigated the mechanisms associated with the low immunogenicity of rAd5 dosed through the p.o. route in mice. The purpose was to identify barriers to effective delivery of rAd vectors to gut tissues and to ascertain sites and strategies for induction of potent cellular and humoral immunity. To investigate the mechanism of the low immunogenicity of rAd vectors through the p.o. route and develop effective delivery of rAd5 and rare serotype rAd35 vectors as gut mucosal HIV vaccines, we have analyzed the obstacles to p.o. immunization, characterized vector transgene expression, and systematically compared immune responses induced by p.o. and local immunization strategies. These studies demonstrated that the higher immune responses were strongly associated with higher gene expression in the intestine and support further study of gut mucosal immunization in SIV challenge models as a potential HIV vaccine strategy.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

Persistence of Transgene Expression Influences CD8+ T-Cell Expansion and Maintenance following Immunization with Recombinant Adenovirus

Previous studies determined that the CD8⁺ T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8⁺ T-cell maintenance following immunization with a recombinant adenovirus.Recombinant human adenovirus 5 (rHuAd5) vector vaccines have garnered considerable attention as platforms for eliciting CD8⁺ T-cell immunity due to their strong immunogenicity in numerous studies, including primate studies and preliminary human trials (30, 32, 53). While these vectors may not represent the optimal serotype for use in humans, due to the high prevalence of preexisting immunity, the robust immunogenicity of rHuAd5 in preclinical models merits further investigation, since the biological information derived from these studies will offer important insights that can be extended to other vaccine platforms.CD8⁺ T cells play an important role in host defense against tumors and viral infections. During the primary phase of the CD8⁺ T-cell response, the activated precursors undergo a rapid and dramatic expansion in cell number, followed by a period of contraction where 80 to 90% of the antigen-specific population dies off, leaving the remaining cells to constitute the memory population (44). CD8⁺ T cells mature over the course of the primary response and acquire the ability to produce gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and, to a lesser degree, interleukin 2 (IL-2). Memory T cells can be divided into central memory and effector memory T cells based on phenotype and anatomical location (44). These phenotypic differences have also been linked to functional differences; however, these relationships remain controversial (2, 16, 20, 46, 55).Various reports have revealed some unexpected qualities of the CD8⁺ T-cell response generated by intramuscular immunization with rHuAd5. The rHuAd5-induced CD8⁺ T-cell response exhibited a protracted contraction phase, and the memory population was composed primarily of effector and effector-memory cells (23, 38, 39, 41, 51). The phenotype of the rHuAd5-elicited CD8⁺ T-cell population was more consistent with the CD8⁺ T-cell population observed in persistent infections, such as polyomavirus (25), murine herpesvirus-68 (35), and murine cytomegalovirus (MCMV) (1) infections, than with that observed in acute infections, such as lymphocytic choriomeningitis virus (LCMV) (44), vaccinia virus (15), and influenza virus (24) infections. Further investigation demonstrated that, as in a persistent infection, antigen presentation persisted for a prolonged period following intramuscular immunization with rHuAd5, and transgene expression could persist at low levels for more than 1 year following infection (41, 51). These data suggest that the sustained effector phenotype may arise from prolonged, low-level transgene expression from the rHuAd5 vector, although this connection was not formally proven. It is difficult to fully appreciate the implications of these observations at this time, since chronic exposure to antigen is often associated with CD8⁺ T-cell dysfunction, yet rHuAd5 vectors have been used successfully to elicit protective immunity in many models of pathogen infection and tumor challenge (5, 54). Nevertheless, other reports have provided evidence that rHuAd5 vectors can, indeed, lead to dysfunctional CD8⁺ T-cell immunity (27, 36). Therefore, further investigation is necessary in order to properly assess the implications of the prolonged antigen expression following rHuAd5 immunization in terms of sustaining a functional memory CD8⁺ T-cell response.In the current report, we sought to determine the relationship between transgene expression and CD8⁺ T-cell maintenance and memory. To this end, we constructed an Ad vector with a doxycycline (DOX)-regulated expression cassette that would permit attenuation of gene expression at various times postinfection. Using this reagent, we addressed two key questions. (i) How does the duration of antigen expression affect the magnitude of primary CD8⁺ T-cell expansion? (ii) Is antigen expression required beyond the peak expansion to maintain the memory CD8⁺ T-cell population?