Promise and problems associated with the use of recombinant AAV for the delivery of anti-HIV antibodies

Abstract

Attempts to elicit antibodies with potent neutralizing activity against a broad range of human immunodeficiency virus (HIV) isolates have so far proven unsuccessful. Long-term delivery of monoclonal antibodies (mAbs) with such activity is a creative alternative that circumvents the need for an immune response and has the potential for creating a long-lasting sterilizing barrier against HIV. This approach is made possible by an incredible array of potent broadly neutralizing antibodies (bnAbs) that have been identified over the last several years. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for long-term delivery for a variety of reasons. The only products made from rAAV are derived from the transgenes that are put into it; as long as those products are not viewed as foreign, expression from muscle tissue may continue for decades. Thus, use of rAAV to achieve long-term delivery of anti-HIV mAbs with potent neutralizing activity against a broad range of HIV-1 isolates is emerging as a promising concept for the prevention or treatment of HIV-1 infection in humans. Experiments in mice and monkeys that have demonstrated protective efficacy against AIDS virus infection have raised hopes for the promise of this approach. However, all published experiments in monkeys have encountered unwanted immune responses to the AAV-delivered antibody, and these immune responses appear to limit the levels of delivered antibody that can be achieved. In this review, we highlight the promise of rAAV-mediated antibody delivery for the prevention or treatment of HIV infection in humans, but we also discuss the obstacles that will need to be understood and solved in order for the promise of this approach to be realized.

Figure 1

Difficulties associated with immune control of HIV infection. The nature of HIV and the evolution of immune evasion strategies of the virus are responsible for why a HIV vaccine has remained an elusive task. HIV preferentially infects and destroys CD4+ T cells (central mediators of the immune systems), especially in the gut-associated lymphoid tissue (GALT). The virus early establishes a reservoir in latently infected CD4+ T cells by integration of proviral DNA into the host cell genome. Recognition by cytotoxic T cells is further exacerbated by downregulation of MHC class I molecules on the surface of virus-infected cells, which is orchestrated by the viral nef gene. Sensing of the pathogenic intruder by the host innate immune system is counteracted by the HIV-1 genes vif and vpu. Antibody and CD8+ T cell responses are readily escaped by selection of antigenic escape variants facilitated by the high mutation rate of the virus. The error-prone reverse transcriptase causes an enormous sequence diversity of the envelope glycoproteins gp120 and gp41 (up to 35% among clades, 20% within clades, 10% in a single infected individual). An extensive glycan shield on the env trimer shields vulnerable targets on envelope (about 50% of the mass of gp120). Abbreviations: reverse transcriptase (RT); integrase (IN); protease (PR); capsid (CA); matrix (MA); nucleocapsid (NC); long terminal repeat (LTR); group-specific antigen (gag); the pol gene encodes RT, IN and PR; viral infectivity factor (Vif); viral protein R (Vpr); viral protein unique (Vpu); negative regulatory factor (Nef); trans-activator of transcription (tat); regulator of expression of virion proteins (rev); envelope (env) gene encodes the glycoprotein gp160 that is processed into gp120 and gp41.

Figure 2

Broadly neutralizing antibodies (bnAbs) to HIV-1. The HIV envelope (env) spike consists of three gp120-gp41 heterodimers that are noncovalently linked to each other. The glycoprotein gp120 harbors the CD4 receptor binding site (CD4bs) and the coreceptor binding site, but the co-receptor binding region is only fully exposed upon binding of gp120 to CD4. The glycoprotein gp41 anchors the env spike into the virus membrane and harbors the fusion machinery that facilitates entry into the target cell. The env trimer spike is considered to be unstable, and decayed or nonfunctional structures appear to be a target for binding/non-neutralizing antibodies. Neutralizing antibodies, especially bnAbs strongly bind the native or functional env trimer spike. Several vulnerable bnAb target sites have been identified and a number of bnAbs bind to at least 5 well-characterized locations on the env trimer. The high-mannose-patch is located on the outer region of gp120, centered on glycans at Asn (N332). bnAbs to this site bind and penetrate the glycan shield and interact with amino acids in the variable loop 3 (V3) of gp120. Apex antibodies bind to lysine-rich regions on the V2 loop, surrounded by glycans at Asn (N160); antibodies to this site require long penetrating heavy chain structures. CD4bs antibodies have structural features that allow binding to the env trimer similar to that of the outer domain of CD4. The CD4bs is protected by the glycan shield and variable loops. MPER-specific antibodies usually have a hydrophobic character due to their binding target that is in close proximity to the lipid bilayer, which is partly recognized by this antibody class. These antibodies are usually self-reactive. Antibodies to the gp120-gp41 interface interact with both glycoproteins and appear to be trimer-specific. Abbreviations: CD4-induced (CD4i), membrane-proximal external region (MPER).

Figure 3

Recombinant adeno-associated virus (rAAV) vectors for the delivery of monoclonal antibodies (mAbs). Wild-type adeno-associated virus (AAV) is a 25 nm small nonenveloped virus that packages a single-stranded DNA genome. The most prominent AAV serotype, AAV2, has a genome size of 4.7 kb and harbors two viral genes (rep and cap) that are flanked by two 145 bp inverted terminal repeats (ITRs). Four Rep proteins (Rep78, Rep68, Rep52, and Rep40) are produced from transcripts using the p5 and p19 promoters, and these proteins are important for viral replication and regulation of AAV gene expression. The virus does not encode a polymerase enzyme and relies on cellular enzymatic activities. Furthermore, AAV relies on the presence of helper viruses such as herpesvirus or adenovirus in order to undergo productive infection (replication, gene expression, and virion production). The cap gene encodes three structural capsid proteins (VP1, VP2, and VP3) from two transcripts using the p40 promoter. For generating recombinant AAV (rAAV), the entire wild-type AAV genome is replaced by a unique transgene cassette (such as for a mAb) flanked by the AAV ITRs, which are the only wild-type sequences remaining. Production of rAAV virions is achieved by triple transfection using the rAAV vector plasmid and two helper plasmids in trans, followed by CsCl purification of the replication-deficient rAAV particles. rAAV particles can be encapsidated by any of the 12 AAV serotypes and more than 100 variants that are available. The conventional single-stranded AAV (ssAAV) vector packages single-stranded DNA. The modified self-complementary AAV (scAAV) encapsidates double-stranded DNA but has only half the packaging capacity of ssAAV. scAAV vectors are produced by modification of the 5’ITR (removal of the terminal resolution site and D sequence). The size limit of the scAAV vector system requires the heavy and light chain sequences of IgG to be placed on separate rAAV vectors.

Figure 4

AAV-mediated gene transfer of anti-HIV monoclonal antibodies (mAbs). A rAAV encoding an anti-HIV mAb is injected into muscle such as the deltoid muscle. Following intramuscular inoculation, rAAV binds to a serotype-specific cellular receptor on myocytes and becomes endocytosed. Virus particles are transported to the nucleus, into which the rAAV genome is released. The single-stranded DNA (in case of ssAAV) is then converted into transcriptionally active double-stranded DNA. Double-stranded rAAV genomes appear to be stabilized by ITR-to-ITR interactions and enzymatic modifications, leading to high molecular weight (MW) rAAV genome polymers that persist in episomal form for the lifetime of the cell. Adult human muscle cells have a lifespan of more than 10 years. The extrachromosomal rAAV DNA forms maintain gene expression and produce the therapeutic mAb, which undergoes the secretory pathway and is released into the circulatory system. Depending on which AAV serotype or variant is used, the injected rAAV virus can also transcytose through multiple cell layers, leading to access to blood vessels. This will transport a proportion of intramuscularly injected rAAV particles to the liver, where cell entry and gene expression will take place. Secretion of an anti-HIV mAb from muscle and liver will potentially create a prophylactic barrier against HIV infection or fight an ongoing HIV infection in a therapeutic setting. AAV, adeno-associated virus; ITR, inverted terminal repeats.