HIV-1 with gag processing defects activates cGAS sensing.

BACKGROUND: Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood. RESULTS: To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS. CONCLUSIONS: These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Prevalence Study of Cellular Capsid-Specific Immune Responses to AAV2, 4, 5, 8, 9, and rh10 in Healthy Donors.

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the United States. Nevertheless, preexisting immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas a preexisting humoral response to AAV capsid is well documented, the prevalence of preexisting capsid-specific T cell responses still needs to be studied and characterized. In this study, we investigated the prevalence of AAV-specific circulating T cells toward AAV2, 4, 5, 8, 9, and rh10 in a large cohort of healthy donors using the standard IFNγ ELISpot assay. We observed the highest prevalence of preexisting cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10, and AAV5 independently of the donors' serological status. An in-depth analysis of T cell responses toward the 2 most prevalent serotypes 8 and 9 shows that IFNγ secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that preexisting immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anticapsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.

Adeno-Associated Viruses (AAV) and Host Immunity - A Race Between the Hare and the Hedgehog.

Adeno-associated viruses (AAV) have emerged as the lead vector in clinical trials and form the basis for several approved gene therapies for human diseases, mainly owing to their ability to sustain robust and long-term in vivo transgene expression, their amenability to genetic engineering of cargo and capsid, as well as their moderate toxicity and immunogenicity. Still, recent reports of fatalities in a clinical trial for a neuromuscular disease, although linked to an exceptionally high vector dose, have raised new caution about the safety of recombinant AAVs. Moreover, concerns linger about the presence of pre-existing anti-AAV antibodies in the human population, which precludes a significant percentage of patients from receiving, and benefitting from, AAV gene therapies. These concerns are exacerbated by observations of cellular immune responses and other adverse events, including detrimental off-target transgene expression in dorsal root ganglia. Here, we provide an update on our knowledge of the immunological and molecular race between AAV (the "hedgehog") and its human host (the "hare"), together with a compendium of state-of-the-art technologies which provide an advantage to AAV and which, thus, promise safer and more broadly applicable AAV gene therapies in the future.

Structural Study of Aavrh.10 Receptor and Antibody Interactions.

Recombinant adeno-associated virus (rAAV) vectors are one of the leading tools for the delivery of therapeutic genes in human gene therapy applications. For a successful transfer of their payload, the AAV vectors have to circumvent potential preexisting neutralizing host antibodies and bind to the receptors of the target cells. Both of these aspects have not been structurally analyzed for AAVrh.10. Here, cryo-electron microscopy and three-dimensional image reconstruction were used to map the binding site of sulfated N-acetyllactosamine (LacNAc; previously shown to bind AAVrh.10) and a series of four monoclonal antibodies (MAbs). LacNAc was found to bind to a pocket located on the side of the 3-fold capsid protrusion that is mostly conserved to AAV9 and equivalent to its galactose-binding site. As a result, AAVrh.10 was also shown to be able to bind to cell surface glycans with terminal galactose. For the antigenic characterization, it was observed that several anti-AAV8 MAbs cross-react with AAVrh.10. The binding sites of these antibodies were mapped to the 3-fold capsid protrusions. Based on these observations, the AAVrh.10 capsid surface was engineered to create variant capsids that escape these antibodies while maintaining infectivity. IMPORTANCE Gene therapy vectors based on adeno-associated virus rhesus isolate 10 (AAVrh.10) have been used in several clinical trials to treat monogenetic diseases. However, compared to other AAV serotypes little is known about receptor binding and antigenicity of the AAVrh.10 capsid. Particularly, preexisting neutralizing antibodies against capsids are an important challenge that can hamper treatment efficiency. This study addresses both topics and identifies critical regions of the AAVrh.10 capsid for receptor and antibody binding. The insights gained were utilized to generate AAVrh.10 variants capable of evading known neutralizing antibodies. The findings of this study could further aid the utilization of AAVrh.10 vectors in clinical trials and help the approval of the subsequent biologics.

Development of a Bispecific Antibody-Based Platform for Retargeting of Capsid Modified AAV Vectors.

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.

Understanding Post Entry Sorting of Adenovirus Capsids; A Chance to Change Vaccine Vector Properties.

Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses' post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.

Overcoming Immunological Challenges Limiting Capsid-Mediated Gene Therapy With Machine Learning.

A key hurdle to making adeno-associated virus (AAV) capsid mediated gene therapy broadly beneficial to all patients is overcoming pre-existing and therapy-induced immune responses to these vectors. Recent advances in high-throughput DNA synthesis, multiplexing and sequencing technologies have accelerated engineering of improved capsid properties such as production yield, packaging efficiency, biodistribution and transduction efficiency. Here we outline how machine learning, advances in viral immunology, and high-throughput measurements can enable engineering of a new generation of de-immunized capsids beyond the antigenic landscape of natural AAVs, towards expanding the therapeutic reach of gene therapy.

Engineered adeno-associated virus 3 vector with reduced reactivity to serum antibodies.

The natural serotypes of adeno-associated virus (AAV) or their variants, such as AAV8 and AAV5, are commonly used as vectors in the clinical programs for liver-targeted gene therapy. While AAV8 vectors are not highly efficient at targeting primary human hepatocytes, AAV3 vectors have recently demonstrated remarkable efficiency at targeting both human and non-human primate hepatocytes. However, the presence of high levels of neutralizing antibodies (NAbs) impedes transduction into hepatocytes, representing a major obstacle to the clinical application of AAV3 vectors. Herein, we engineered the viral capsid to reduce its reactivity with pre-existing NAbs, thereby enhancing the transduction efficiency. By introducing three substitutions (S472A, S587A, and N706A) on the surface loop of AAV3B capsid protein, we generated a triple mutant AAV3 (AAV.GT5) vector with less reactivity to anti-AAV capsid NAbs. While the transduction efficiency of AAV.GT5 into human hepatocellular cell lines was similar to those of parental AAV3B, it was 50-fold higher for hepatocytes derived from humanized mice compared to AAV8 vectors. Moreover, the AAV.GT5 vector yield was similar to those of the AAV2 and AAV3B vectors. Thus, high resistance to pre-existing NAbs makes AAV.GT5 a promising candidate for future liver-targeted gene therapy clinical trials.

From Capsids to Complexes: Expanding the Role of TRIM5α in the Restriction of Divergent RNA Viruses and Elements.

An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses.

Capsid and Genome Modification Strategies to Reduce the Immunogenicity of Adenoviral Vectors.

Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.

Parvovirus Capsid-Antibody Complex Structures Reveal Conservation of Antigenic Epitopes Across the Family.

The parvoviruses are small nonenveloped single stranded DNA viruses that constitute members that range from apathogenic to pathogenic in humans and animals. The infection with a parvovirus results in the generation of antibodies against the viral capsid by the host immune system to eliminate the virus and to prevent re-infection. For members currently either being developed as delivery vectors for gene therapy applications or as oncolytic biologics for tumor therapy, efforts are aimed at combating the detrimental effects of pre-existing or post-treatment antibodies that can eliminate therapeutic benefits. Therefore, understanding antigenic epitopes of parvoviruses can provide crucial information for the development of vaccination applications and engineering novel capsids able to escape antibody recognition. This review aims to capture the information for the binding regions of ∼30 capsid-antibody complex structures of different parvovirus capsids determined to date by cryo-electron microscopy and three-dimensional image reconstruction. The comparison of all complex structures revealed the conservation of antigenic regions among parvoviruses from different genera despite low sequence identity and indicates that the available data can be used across the family for vaccine development and capsid engineering.

JCPyV VP1 Mutations in Progressive MultifocalLeukoencephalopathy: Altering Tropismor Mediating Immune Evasion?

Polyomaviruses are ubiquitous human pathogens that cause lifelong, asymptomatic infections in healthy individuals. Although these viruses are restrained by an intact immune system, immunocompromised individuals are at risk for developing severe diseases driven by resurgent viral replication. In particular, loss of immune control over JC polyomavirus can lead to the development of the demyelinating brain disease progressive multifocal leukoencephalopathy (PML). Viral isolates from PML patients frequently carry point mutations in the major capsid protein, VP1, which mediates virion binding to cellular glycan receptors. Because polyomaviruses are non-enveloped, VP1 is also the target of the host's neutralizing antibody response. Thus, VP1 mutations could affect tropism and/or recognition by polyomavirus-specific antibodies. How these mutations predispose susceptible individuals to PML and other JCPyV-associated CNS diseases remains to be fully elucidated. Here, we review the current understanding of polyomavirus capsid mutations and their effects on viral tropism, immune evasion, and virulence.

Antibody escape by polyomavirus capsid mutation facilitates neurovirulence.

JCPyV polyomavirus, a member of the human virome, causes progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease in individuals receiving immunomodulatory therapies. Mutations in the major viral capsid protein, VP1, are common in JCPyV from PML patients (JCPyV-PML) but whether they confer neurovirulence or escape from virus-neutralizing antibody (nAb) in vivo is unknown. A mouse polyomavirus (MuPyV) with a sequence-equivalent JCPyV-PML VP1 mutation replicated poorly in the kidney, a major reservoir for JCPyV persistence, but retained the CNS infectivity, cell tropism, and neuropathology of the parental virus. This mutation rendered MuPyV resistant to a monoclonal Ab (mAb), whose specificity overlapped the endogenous anti-VP1 response. Using cryo-EM and a custom sub-particle refinement approach, we resolved an MuPyV:Fab complex map to 3.2 Å resolution. The structure revealed the mechanism of mAb evasion. Our findings demonstrate convergence between nAb evasion and CNS neurovirulence in vivo by a frequent JCPyV-PML VP1 mutation.

Coevolution of Adeno-associated Virus Capsid Antigenicity and Tropism through a Structure-Guided Approach.

Adeno-associated viruses (AAV) are composed of nonenveloped, icosahedral protein shells that can be adapted to package and deliver recombinant therapeutic DNA. Approaches to engineer recombinant capsids for gene therapy applications have focused on rational design or library-based approaches that can address one or two desirable attributes; however, there is an unmet need to comprehensively improve AAV vector properties. Such cannot be achieved by utilizing sequence data alone but requires harnessing the three-dimensional (3D) structural properties of AAV capsids. Here, we solve the structures of a natural AAV isolate complexed with antibodies using cryo-electron microscopy and harness this structural information to engineer AAV capsid libraries through saturation mutagenesis of different antigenic footprints. Each surface loop was evolved by infectious cycling in the presence of a helper adenovirus to yield a new AAV variant that then serves as a template for evolving the next surface loop. This stepwise process yielded a humanized AAV8 capsid (AAVhum.8) displaying nonnatural surface loops that simultaneously display tropism for human hepatocytes, increased gene transfer efficiency, and neutralizing antibody evasion. Specifically, AAVhum.8 can better evade neutralizing antisera from multiple species than AAV8. Further, AAVhum.8 displays robust transduction in a human liver xenograft mouse model with expanded tropism for both murine and human hepatocytes. This work supports the hypothesis that critical properties, such as AAV capsid antibody evasion and tropism, can be coevolved by combining rational design and library-based evolution for clinical gene therapy.IMPORTANCE Clinical gene therapy with recombinant AAV vectors has largely relied on natural capsid isolates. There is an unmet need to comprehensively improve AAV tissue tropism, transduction efficiency, and antibody evasion. Such cannot be achieved by utilizing capsid sequence data alone but requires harnessing the 3D structural properties of AAV capsids. Here, we combine rational design and library-based evolution to coevolve multiple, desirable properties onto AAV by harnessing 3D structural information.

Induction of adaptive immune responses against antigens incorporated within the capsid of simian virus 40.

Simian virus 40 (SV40) is a monkey polyomavirus. The capsid structure is icosahedral and comprises VP1 units that measure 45 nm in diameter. Five SV40 VP1 molecules form one pentamer subunit, and a single icosahedral subunit comprises 72 pentamers; a single SV40 VP1 capsid comprises 360 SV40 VP1 molecules. In a previous study, we showed that an influenza A virus matrix protein 1 (M1) CTL epitope inserted within SV40 virus-like particles (VLPs) induced cytotoxic T lymphocytes (CTLs) without the need for an adjuvant. Here, to address whether SV40 VLPs induce adaptive immune responses against VLP-incorporated antigens, we prepared SV40 VLPs containing M1 or chicken ovalbumin (OVA). This was done by fusing M1 or OVA with the carboxyl terminus of SV40 VP2 and co-expressing them with SV40 VP1 in insect cells using a baculovirus vector. Intraperitoneal (i.p.) or intranasal administration of SV40 VLPs incorporating M1 induced the production of CTLs specific for the M1 epitope without the requirement for adjuvant. The production of antibodies against SV40 VLPs was also induced by i.p. administration of SV40 VLPs in the absence of adjuvant. Finally, the administration of SV40 VLPs incorporating OVA induced anti-OVA antibodies in the absence of adjuvant; in addition, the level of antibody production was comparable with that after i.p. administration of OVA plus alum adjuvant. These results suggest that the SV40 capsid incorporating foreign antigens can be used as a vaccine platform to induce adaptive immune responses without the need for adjuvant.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 1010 to 2 × 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies.

Neutralizing antibodies to adeno-associated virus (AAV) vectors are highly prevalent in humans1,2, and block liver transduction3-5 and vector readministration6; thus, they represent a major limitation to in vivo gene therapy. Strategies aimed at overcoming anti-AAV antibodies are being studied7, which often involve immunosuppression and are not efficient in removing pre-existing antibodies. Imlifidase (IdeS) is an endopeptidase able to degrade circulating IgG that is currently being tested in transplant patients8. Here, we studied if IdeS could eliminate anti-AAV antibodies in the context of gene therapy. We showed efficient cleavage of pooled human IgG (intravenous Ig) in vitro upon endopeptidase treatment. In mice passively immunized with intravenous Ig, IdeS administration decreased anti-AAV antibodies and enabled efficient liver gene transfer. The approach was scaled up to nonhuman primates, a natural host for wild-type AAV. IdeS treatment before AAV vector infusion was safe and resulted in enhanced liver transduction, even in the setting of vector readministration. Finally, IdeS reduced anti-AAV antibody levels from human plasma samples in vitro, including plasma from prospective gene therapy trial participants. These results provide a potential solution to overcome pre-existing antibodies to AAV-based gene therapy.

Correlation between cervical HPV DNA detection and HPV16 seroreactivity measured with L1-only and L1+L2 viral capsid antigens.

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.

Evading the AAV Immune Response in Mucopolysaccharidoses.

The humoral immune response elicited by adeno-associated virus (AAV)-mediated gene therapy for the treatment of mucopolysaccharidoses (MPS) poses a significant challenge to achieving therapeutic levels of transgene expression. Antibodies targeting the AAV capsid as well as the transgene product diminish the production of glycosaminoglycan (GAG)-degrading enzymes essential for the treatment of MPS. Patients who have antibodies against AAV capsid increase in number with age, serotype, and racial background and are excluded from the clinical trials at present. In addition, patients who have undergone AAV gene therapy are often excluded from the additional AAV gene therapy with the same serotype, since their acquired immune response (antibody) against AAV will limit further efficacy of treatment. Several methods are being developed to overcome this immune response, such as novel serotype design, antibody reduction by plasmapheresis and immunosuppression, and antibody evasion using empty capsids and enveloped AAV vectors. In this review, we examine the mechanisms of the anti-AAV humoral immune response and evaluate the strengths and weaknesses of current evasion strategies in order to provide an evidence-based recommendation on evading the immune response for future AAV-mediated gene therapies for MPS.