High concordance of ELISA and neutralization assays allows for the detection of antibodies to individual AAV serotypes.

Prescreening of participants in clinical trials that use adeno-associated virus (AAV) vectors is required to identify naive participants, as preexisting neutralizing antibodies can limit the efficacy of AAV gene therapies. The presence of antibodies to individual AAV serotypes is typically detected by neutralization assay. To streamline the screening process, we compared an ELISA-based screening method with a neutralization assay for the detection of antibodies against AAV1, AAV8, and AAV9 in a collection of 50 rhesus macaque sera and 20 human sera. We observed a high level of concordance between the two assays (Pearson r > 0.8) for all three serotypes in both sample sets. We thus investigated pre- vs post-vector inoculation sera samples from rhesus macaques that received AAV1 or AAV8 vector inoculations for cross-reactive anti-AAV antibodies. All 12 macaques seroconverted to the vector they received, but many also reacted to the other serotypes. Our results validate an easy-to-use ELISA for reliable detection of antibodies to individual serotypes of AAV. Our results also demonstrate that an antibody response post-AAV inoculation may partially cross-react with other AAV serotypes. Overall, these results suggest that either assay can be used by academic labs for prescreening samples for preexisting anti-AAV antibodies.

Glycoengineering of AAV-delivered monoclonal antibodies yields increased ADCC activity.

The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo, current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies.

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV.

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240-350 µg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Induction of Transient Virus Replication Facilitates Antigen-Independent Isolation of SIV-Specific Monoclonal Antibodies.

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8ß-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo.

Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys.

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody.

Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity.

Recombinant adeno-associated virus (rAAV)-mediated gene delivery can efficiently target muscle tissues to serve as "biofactories" for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent anti-transgene immunity is therefore crucial. The employment of endogenous microRNA (miRNA)-mediated regulation to detarget transgene expression from antigen presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of anti-transgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142 binding sites efficiently repress co-stimulatory signals in dendritic cells, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.

Adeno-Associated Virus Delivery of Anti-HIV Monoclonal Antibodies Can Drive Long-Term Virologic Suppression.

Long-term delivery of anti-HIV monoclonal antibodies (mAbs) using adeno-associated virus (AAV) vectors holds promise for the prevention and treatment of HIV infection. We describe a therapy trial in which four rhesus monkeys were infected with SHIV-AD8 for 86 weeks before receiving the AAV-encoded mAbs 3BNC117, 10-1074, and 10E8. Although anti-drug antibody (ADA) responses restricted mAb delivery, one monkey successfully maintained 50-150 µg/mL of 3BNC117 and 10-1074 for over 2 years. Delivery of these two mAbs to this monkey resulted in an abrupt decline in plasma viremia, which remained undetectable for 38 successive measurements over 3 years. We generated two more examples of virologic suppression using AAV delivery of a cocktail of four mAbs in a 12-monkey study. Our results provide proof of concept for AAV-delivered mAbs to produce a "functional cure." However, they also serve as a warning that ADAs may be a problem for practical application of this approach in humans.

Anti-drug Antibody Responses Impair Prophylaxis Mediated by AAV-Delivered HIV-1 Broadly Neutralizing Antibodies.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs.

Potent Plasmablast-Derived Antibodies Elicited by the National Institutes of Health Dengue Vaccine.

Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes develops only after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long-sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. In this study, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan Institute. Eleven days after vaccination, we observed an ∼70-fold expansion of the plasmablast population. We generated 21 monoclonal Abs (MAbs) from singly sorted plasmablasts. These MAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen MAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 µg/ml or less; 6 of the 21 MAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing MAbs (nMAbs). Furthermore, the P3D05 nMAb neutralized DENV3 with extraordinary potency (concentration to achieve half-maximal neutralization [Neut50] = 0.03 µg/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that preexisting responses elicited by a previous DENV3 infection were recalled by immunization.IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut50 < 0.1 µg/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in phase III trials.

Dengue Virus Evades AAV-Mediated Neutralizing Antibody Prophylaxis in Rhesus Monkeys.

Development of vaccines against mosquito-borne Flaviviruses is complicated by the occurrence of antibody-dependent enhancement (ADE), which can increase disease severity. Long-term delivery of neutralizing antibodies (nAbs) has the potential to effectively block infection and represents an alternative to vaccination. The risk of ADE may be avoided by using prophylactic nAbs harboring amino acid mutations L234A and L235A (LALA) in the immunoglobulin G (IgG) constant region. Here, we used recombinant adeno-associated viruses (rAAVs) to deliver the anti-dengue virus 3 (DENV3) nAb P3D05. While the administration of rAAV-P3D05-rhesus immunoglobulin G1 (rhIgG1)-LALA to rhesus macaques engendered DENV3-neutralizing activity in serum, it did not prevent infection. The emergence of viremia following DENV3 challenge was delayed by 3-6 days in the rAAV-treated group, and replicating virus contained the envelope mutation K64R. This neutralization-resistant variant was also confirmed by virus outgrowth experiments in vitro. By delivering P3D05 with unmutated Fc sequences, we further demonstrated that DENV3 also evaded wild-type nAb prophylaxis, and serum viral loads appeared to be higher in the presence of low levels of unmutated P3D05-rhIgG1. Our study shows that a vectored approach for long-term delivery of nAbs with the LALA mutations is promising, but prophylaxis using a single nAb is likely insufficient at preventing DENV infection and replication.

A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus.

The isolation of neutralizing monoclonal antibodies (nmAbs) against the Zika virus (ZIKV) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. Here we describe the characterization of human mAbs from the plasmablasts of an acutely infected patient. One of the 18 mAbs had the unusual feature of binding to and neutralizing ZIKV despite not appearing to have been diversified by affinity maturation. This mAb neutralized ZIKV (Neut50 ~ 2 µg/ml) but did not react with any of the four dengue virus serotypes. Except for the expected junctional diversity created by the joining of the V-(D)-J genes, there was no deviation from immunoglobulin germline genes. This is a rare example of a human mAb with neutralizing activity in the absence of detectable somatic hypermutation. Importantly, binding of this mAb to ZIKV was specifically inhibited by human plasma from ZIKV-exposed individuals, suggesting that it may be of value in a diagnostic setting.

Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG.

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

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.

Host Anti-antibody Responses Following Adeno-associated Virus-mediated Delivery of Antibodies Against HIV and SIV in Rhesus Monkeys.

Long-term delivery of antibodies against the human immunodeficiency virus (HIV) using adeno-associated virus (AAV) vectors is a promising approach for the prevention or treatment of HIV infection. However, host antibody responses to the delivered antibody are a serious concern that could significantly limit the applicability of this approach. Here, we describe the dynamics and characteristics of the anti-antibody responses in monkeys that received either rhesus anti-simian immunodeficiency virus (SIV) antibodies (4L6 or 5L7) in prevention trials or a combination of rhesusized human anti-HIV antibodies (1NC9/8ANC195/3BNC117 or 10-1074/10E8/3BNC117) in therapy trials, all employing AAV1 delivery of IgG1. Eight out of eight monkeys that received the anti-HIV antibodies made persisting antibody responses to all three antibodies in the mix. Six out of six uninfected monkeys that received the anti-SIV antibody 4L6 and three out of six of those receiving anti-SIV antibody 5L7 also generated anti-antibodies. Both heavy and light chains were targeted, predominantly or exclusively to variable regions, and reactivity to complementarity-determining region (CDR)-H3 peptide could be demonstrated. There was a highly significant correlation of the magnitude of anti-antibody responses with the degree of sequence divergence of the delivered antibody from germline. Our results suggest the need for effective strategies to counteract the problem of antibody responses to AAV-delivered antibodies.

AAV-Delivered Antibody Mediates Significant Protective Effects against SIVmac239 Challenge in the Absence of Neutralizing Activity.

Long-term delivery of potent broadly-neutralizing antibodies is a promising approach for the prevention of HIV-1 infection. We used AAV vector intramuscularly to deliver anti-SIV monoclonal antibodies (mAbs) in IgG1 form to rhesus monkeys. Persisting levels of delivered mAb as high as 270 µg/ml were achieved. However, host antibody responses to the delivered antibody were observed in 9 of the 12 monkeys and these appeared to limit the concentration of delivered antibody that could be achieved. This is reflected in the wide range of delivered mAb concentrations that were achieved: 1-270 µg/ml. Following repeated, marginal dose, intravenous challenge with the difficult-to-neutralize SIVmac239, the six monkeys in the AAV-5L7 IgG1 mAb group showed clear protective effects despite the absence of detectable neutralizing activity against the challenge virus. The protective effects included: lowering of viral load at peak height; lowering of viral load at set point; delay in the time to peak viral load from the time of the infectious virus exposure. All of these effects were statistically significant. In addition, the monkey with the highest level of delivered 5L7 mAb completely resisted six successive SIVmac239 i.v. challenges, including a final challenge with a dose of 10 i.v. infectious units. Our results demonstrate the continued promise of this approach for the prevention of HIV-1 infection in people. However, the problem of anti-antibody responses will need to be understood and overcome for the promise of this approach to be effectively realized.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.