Protection against SIV in Rhesus Macaques Using Albumin and CD4-Based Vector-Mediated Gene Transfer.

Antibody-like molecules were evaluated with potent simian immunodeficiency virus (SIV) neutralizing properties (immunoadhesins) that were delivered by a recombinant adeno-associated virus (rAAV) vector in the SIV-infected rhesus macaque model. When injected intramuscularly into the host, the vector directs in vivo production of the transgenes with antibody-like binding properties that lead to serum neutralizing activity against SIV. To extend the half-life of the immunoadhesins, rhesus cluster of differentiation 4 (CD4) and a single-chain antibody (4L6) were fused with albumin molecules, and these constructs were tested in our model of SIV infection. Antibody-based immunoadhesins provided high serum neutralizing titers against the original SIV strain. CD4-based immunoadhesins provided a wider spectrum of neutralization against different SIV strains in comparison to antibody-based therapeutics and had the potential to protect against high viral challenging doses. Although the albumin-antibody fusion immunoadhesin provided strong and prolonged protection of the immunized animals against SIV challenge, the albumin-CD4 fusion altered the specificity and decreased the overall protection effectiveness of the immunoadhesin in comparison to the antibody-based molecules. Albumin-based immunoadhesins increase in vivo longevity of the immune protection; however, they present challenges likely linked to the induction of anti-immunoadhesin antibodies.

Engineered Expression of Broadly Neutralizing Antibodies Against Human Immunodeficiency Virus.

This review discusses recent progress made in developing a vaccine and novel treatments for human immunodeficiency virus (HIV). It highlights the shortcomings of the RV144 vaccination trial [ALVAC-HIV (vCP1521) and AIDSVAX B/E] and the current standard of care and proposes that engineered expression of broadly neutralizing antibodies (bNAbs) against HIV-1 could overcome these shortcomings. Current developments in three major lines of research on HIV prevention and treatment using bNAbs are reviewed: firstly, the use of sequential immunogens to activate B cells to express bNAbs; secondly, the delivery of novel and extremely potent bNAbs through passive administration; and finally, the use of gene transfer using adeno-associated viral vectors to deliver bNAbs.

Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle.

Gene augmentation therapy as a strategy to treat alpha-1 antitrypsin (AAT) deficiency has reached phase 2 clinical testing in humans. Sustained serum levels of AAT have been observed beyond one year after intramuscular administration of a recombinant adeno-associated virus (rAAV) vector expressing the AAT gene. In this study, sequential muscle biopsies obtained at 3 and 12 months after vector injection were examined for the presence of rAAV vector genomes. Each biopsy sample contained readily detectable vector DNA, the majority of which existed as double-stranded supercoiled and open circular episomes. Episomes persisted through 12 months, although at slightly lower levels than observed at 3 months. There was a clear dose response when comparing the low- and mid-vector-dose groups to the high-dose group. The highest absolute copy numbers were found in a high-dose subject, and serum AAT levels at 12 months confirmed that the high-dose group also had the highest sustained serum AAT levels. Sequence analysis revealed that the vast majority of episomes contained double-D inverted terminal repeats ranging from fully intact to severely deleted. Molecular clones of vector genomes derived directly from the biopsies were transcriptionally active, potentially identifying them as the source of serum AAT in the trial subjects.

Vector-mediated antibody gene transfer for infectious diseases.

This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

Adeno-associated virus delivery of broadly neutralizing antibodies.

PURPOSE OF REVIEW: In the present review, we will discuss the emerging field of vector-mediated antibody gene transfer as an alternative HIV vaccine. This approach is an improvement over classical passive immunization strategies that administer antibodies to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, resulting in long-term endogenous antibody expression from the injected muscle that confers protective immunity. RECENT FINDINGS: Large numbers of very potent and broadly neutralizing HIV antibodies have recently been isolated and characterized. Vector-mediated antibody gene transfer allows one to immediately use these antibodies as a vaccine. Gene transfer studies in both mice and monkeys demonstrate long-term antibody expression in serum from a single injection at concentrations that provide sterilizing immunity. SUMMARY: Vector-mediated antibody gene transfer can rapidly move existing, potent anti-HIV molecules into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown little promise.

Vector-Mediated In Vivo Antibody Expression.

This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

Gene-based vaccine approaches for respiratory syncytial virus.

A respiratory syncytial virus (RSV) vaccine has remained elusive for decades, largely due to the failure of a formalin-inactivated RSV vaccine in the 1960s that resulted in enhanced disease upon RSV exposure in the immunized individuals. Vaccine development has also been hindered by the incomplete immunity conferred by natural infection allowing for re-infection at any time, and the immature immune system and circulating maternal antibodies present in the neonate, the primary target for a vaccine. This chapter will review the use of gene delivery, both nonviral and viral, as a potential vaccine approach for human RSV. Many of these gene-based vaccines vectors elicit protective immune responses in animal models. None of the RSV gene-based platforms have progressed into clinical trials, mostly due to uncertainty regarding the direct translation of animal model results to humans and the hesitancy to invest in costly clinical trials with the potential for unclear and complicated immune responses. The continued development of RSV vaccine gene-based approaches is warranted because of their inherent flexibility with regard to composition and administration. It is likely that multiple candidate vaccines will reach human testing in the next few years.

A novel self-replicating chimeric lentivirus-like particle.

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

A phase 2 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 vaccine based on adeno-associated virus.

The recombinant vaccine, tgAAC09, based on an adeno-associated virus serotype 2 (AAV2) vector encoding HIV-1 subtype C Gag, protease, and part of reverse transcriptase, induced robust T cell and antibody responses in nonhuman primates. In a previous phase I study in 80 healthy HIV-seronegative European and Indian adults, the vaccine was generally safe, well tolerated, and modestly immunogenic when administered once at doses up to 3 x 10(11) DRP. This phase II double-blind, randomized, placebo-controlled trial tested two administrations and a higher dosage of tgAAC009. Ninety-one healthy HIV-seronegative adults from three African countries were given one of three dosage levels of tgAAC09 (3 x 10(10), 3 x 10(11), or 3 x 10(12) DRP) intramuscularly, either at a 6- or 12-month interval; follow-up was 18 months. Overall, 65% and 57% of vaccine recipients experienced local and systemic signs and symptoms, respectively, most being mild. Frequency and severity were not dose related and were similar to those in placebo recipients. No vaccine-related serious adverse events were reported. Overall, HIV-specific T cell responses were detected by IFN-gamma ELISPOT in 17/69 (25%) vaccine recipients with 38% (10/26) responders in the highest dosage group. The response rate improved significantly with boosting at 6, but not 12 months, in the 3 x 10(11) and 3 x 10(12) dosage groups only. Neutralizing antibody titers to the AAV2 did not alter the frequency of immune responses to HIV. Two doses of tgAAC09 were well tolerated at the dosage levels given. Fewer than half the recipients of the highest vaccine dosage, 3 x 10(12) DRP, had T cell responses to HIV.

Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys.

The key to an effective HIV vaccine is development of an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the simian immunodeficiency virus (SIV) model, we have taken a markedly different approach: delivery to muscle of an adeno-associated virus gene transfer vector expressing antibodies or antibody-like immunoadhesins having predetermined SIV specificity. With this approach, SIV-specific molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and have observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds considerable promise as a unique approach to an effective HIV vaccine.

Genetic vaccines for anthrax based on recombinant adeno-associated virus vectors.

Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments encoding activated PA (PA63), or LF, were synthesized and cloned into optimized rAAV1 vectors containing a human cytomegalovirus (hCMV) promoter and synthetic optimized leader. Serum from rabbits immunized intramuscularly with rAAV1/PA (monovalent), rAAV1/LF (monovalent), rAAV1/PA + rAAV1/LF (bivalent), or rAAV1/enhanced green fluorescent protein (control) exhibited substantial PA- and LF-specific antibody responses at 4 weeks by both western blot (> 1:10,000 dilution) and enzyme-linked immunosorbent assay (ELISA) (mean end-point titer: 32,000-260,000), and contained anthrax LeTx-neutralizing activity in vitro, with peak titers approximating those of a rabbit hyperimmune antisera raised against soluble PA and LF. Compared to the monovalent groups (rAAV1/PA or rAAV1/LF), the bivalent group (rAAV1/PA + rAAV1/LF) exhibited marginally higher ELISA and neutralization activity with dual specificity for both PA and LF. The finding of robust neutralizing antibody responses after a single injection of these rAAV1-based vectors supports their further development as candidate anthrax vaccines.

Infectious molecular clones of adeno-associated virus isolated directly from human tissues.

Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine.

A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman primates. In this randomized, dose escalation phase I trial, HIV-uninfected healthy volunteers (50 in Europe, 30 in India) received a single intramuscular injection of tgAAC09 at 3 x 10(9) DNase resistant particles (DRP) (n = 16), 3 x 10(10) DRP (n = 23), 3 x 10(11) DRP (n = 25), or placebo (n = 16). Twenty-one participants in Europe received a second (boost) dose of 3 x 10(11) DRP tgAAC09 or placebo at least 24 weeks after the first injection. The vaccine was safe and well-tolerated after initial and boost vaccination. Local and systemic reactogenicity was experienced by 13-25% of participants and was not dose related. No vaccine-related serious adverse events were reported. Modest HIV-specific T cell responses were detected in 7/64 vaccinees (40-385 SFC/10(6) PBMC), with 16% (4/25) responders in the highest dose group. All responses were to Gag epitopes. tgAAC09 appears to be safe, well-tolerated, and modestly immunogenic. Further evaluation of higher doses of tgAAC09 and boost injections is ongoing in Africa.

Dynamics of simian immunodeficiency virus populations in blood and cerebrospinal fluid over the full course of infection.

UNLABELLED: BACKGROUND. Human immunodeficiency virus (HIV) replication and compartmentalization in the central nervous system, including in cerebrospinal fluid (CSF), are associated with severe neurological disease and may contribute to viral persistence during antiretroviral therapy. To understand the relationships between viral populations in multiple compartments, we performed a systematic longitudinal characterization of viral populations in blood plasma and CSF obtained at short time intervals over the full course of infection in 3 macaques infected with simian immunodeficiency virus (SIVsm strain E660). METHODS: Complex viral genetic populations in blood plasma and CSF were characterized using a heteroduplex tracking assay targeted to the V1/V2 hypervariable region of env. To identify signs of neurological disease, monocyte chemoattractant protein (MCP)-1 levels in CSF and CD68(+) monocyte/macrophage infiltration in brain tissues were quantified. RESULTS: Two patterns of blood/CSF viral dynamics were apparent as infection progressed: concordant blood/CSF viral evolution and discordant blood/CSF viral evolution. Perivascular CD68(+) cells in autopsy brain tissue and elevated CSF MCP-1 levels accompanied blood/CSF viral population discordance but not concordance. CONCLUSIONS: Two distinct patterns of blood/CSF viral population dynamics can be observed in SIV-infected macaques, and the patterns may be associated with different neurological disease outcomes.

Characterization of adeno-associated virus genomes isolated from human tissues.

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

Molecular characterization of adeno-associated viruses infecting children.

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

Novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques.

Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.

Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies.

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infected Macaca mulatta macaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P = 0.05). Macaques that maintained

Correlation between env V1/V2 region diversification and neutralizing antibodies during primary infection by simian immunodeficiency virus sm in rhesus macaques.

Evolution of the domain encoding the V1/V2 variable region of the simian immunodeficiency virus sm (SIVsm) envelope (env) gene was analyzed in relation to route of virus challenge, virus load, and neutralizing antibody (NAb) titers during primary infection of rhesus macaques with the pathogenic SIVsmE660 isolate. In this model system animals are initially infected with multiple viruses as evidenced by the presence of multiple V1/V2 genotypic variants that could be resolved by using a heteroduplex tracking assay (HTA). Overlapping subsets of the multiple variants were established in each animal. There was no selection for the establishment of specific variants in comparing intravenous- and intrarectal-challenged macaques at week 2 postinfection, suggesting that no genotypic selection occurred at the mucosal surface. There was an initial period of significant stability of the V1/V2 variants. Macaques challenged intravenously displayed subsequent V1/V2 diversification significantly earlier than macaques challenged intrarectally and well past the initial resolution of viremia. The time when SIVsmE660-specific NAbs reached a threshold titer of 100 was significantly correlated with the timing of V1/V2 diversification, even though antibodies to the Env protein could be detected much earlier. The time when NAbs reached a titer of 400 was significantly correlated with virus load late in infection. These results show that the route of infection affects the timing of V1/V2 diversification and that this diversification is correlated with the maturation of a specific NAb response. However, prior immunization capable of priming an anamnestic Env antibody response did not accelerate V1/V2 diversification. This result suggests that diversification of the SIV env V1/V2 region is the result of a type-specific antibody response.