June 2011 TreatmentISSUES Covalent vaccination and catalytic antibodies: A new way of looking at an HIV vaccine

Authors: Sudhir Paul,*† Stephanie A. Planque,* Yasuhiro the viral coat proteins. The mutable coat regions are also Nishiyama,* Miguel Escobar,* Zachary Barnett‡ and its dominant antigenic regions, also known as epitopes, Richard J. Massey† against which the immune system produces antibodies and T lymphocytes.2 The original infecting strain induces Introduction a robust immune response, but new quasi-strains develop There is global concern regarding HIV drug-resistance, over the course of infection, and protection against the virus drug toxicity, and increasing drug costs. Many health care is transient at best. Similarly, the antigenic constituents professionals believe that eradicating HIV will require incorporated in previously-tested candidate vaccines were development of a vaccine that prevents infection by the drawn from a single HIV strain or at most a few strains. virus. Yet, one by one, classical vaccine approaches used The candidate vaccines induced antibody responses and for combating other infections have proved ineffective for T cell responses mostly directed to the mutable coat pro- HIV in clinical trials. tein regions, compromising their efficacy against structur- Nearly three decades of research has been invested ally divergent virus strains in different individuals and in in understanding how HIV overcomes immune defenses different parts of the world. and why candidate HIV vaccines have been ineffective. Over the course of the humoral immune response, anti- Immune defense is provided by an ensemble of molecules body complementarity determining regions (CDRs) undergo and cells with innate and adaptive capability to counter rapid mutations under the selective pressure of antigen infectious microbes. The innate immune capabilities have binding. This process generally generates neutralizing anti- evolved over millions of years of evolution. Their func- bodies capable of high affinity antigen binding. One of the tional importance resides in the immediate blockade of few immune vulnerabilities of HIV is the maintenance of infection, for example, killing of microbes by macrophages its exposed CD4 binding site (CD4BS) on the surface of the that migrate to the infection site due to an inflammatory coat protein gp120 in mostly constant form. The CD4BS is response. Vaccines generally work by inducing adaptive essential for virus-host cell binding and infection. Despite immunity developed over days to weeks. The functional minimal chemical variability of the CD4BS, the immune mediators of adaptive immunity are antibodies and T lym- system fails to mount a sufficiently protective antibody phocytes that specifically recognize microbial antigens response to the CD4BS. and help remove the microbe. HIV has developed various The reasons are complex. First, individual epitopes mechanisms to overcome natural immune defenses of within the CD4BS are conformationally plastic, that is, the humans. These mechanisms are also the reason for the three-dimensional epitope structure can change during failure of classical immunological approaches to yield an the process of infection. Initial CD4 binding at the CD4BS effective HIV vaccine. region located in the outer gp120 domain (CD4BSod) may induce a conformational change of the CD4BS core region Challenges posed by HIV immune properties composed of amino acids 421-433 (CD4BScore) that is Thousands of different HIV-1 strains have emerged. Most essential for stable HIV-host cell binding. Consequently, infections are initiated by strains that utilize chemokine the CD4BScore might exist in a conformation vulnerable coreceptor CCR5 for entry into host cells. Coreceptor to immune attack only transiently during the process of CXCR4-dependent strains emerge with time. Both types CD4BS-CD4 binding. Second, HIV utilizes an unusual evolu- of strains use CD4 as the primary host receptor to infect tionary trick to preclude production of a protective antibody T cells and macrophages. Different parts of the world are response by B lymphocytes. The CD4BScore expresses dominated by strains belonging to different HIV-1 subtypes.1 superantigen character.3,4 Superantigens bind specifically Subtype C strains are found primarily in the developing to innately-produced antibodies expressed on the surface world and account for a majority of infections globally. of B lymphocytes, the B cell receptors. Unlike the stimula- A central problem is that most exposed components tory binding of traditional antigens to the B cell receptor, of HIV mutate rapidly, generating structural variations of superantigen binding occurs at the antibody framework regions, and the functional consequence is down-regulation tested vaccine candidates. Burton and coworkers have of B cell differentiation, premature cell death and failure to identified rare antibodies that recognize a segment of the mount an adaptive antibody response. We suggested that CD4BS (the CD4BSod) and neutralize genetically divergent the innate superantigen recognition capability of antibodies HIV strains comparatively broadly.10 Reverse-engineering was originally developed by Darwinian evolution process- of peptides with structure complementary to the neu- es over millions of years as a defense against primordial tralizing antibody binding site can be conceived as a microbes.5 HIV appears to have evolved a CD4BS with route to a vaccine that induces the synthesis of similar superantigenic character as the means to preclude an neutralizing antibodies upon administration to humans. adaptive antibody response. A peptide immunogen designed using as template a neutralizing antibody to a segment of the CD4BS did not Novel vaccine approaches induce broadly neutralizing antibodies.11 Targeting a larger Induction of neutralizing antibodies is the cornerstone CD4BS surface area by a reverse-engineered immunogen of effective vaccination. Following failure of candidate could be more fruitful. protein and polypeptide vaccines to induce sufficient neu- Our studies have identified the CD4BScore as the prover- tralizing antibodies to the free virus,6 the focus shifted to bial Achilles heel of the virus. In the rare circumstances that developing candidate DNA vaccines that induce cytotoxic anti-CD4BScore antibodies are produced, they neutralize HIV T cells directed to HIV infected cells.7 This approach was strains from across the world with exceptional potency.12,13 also ineffective. The RV144 vaccine composed of full- Such antibodies were found in non-infected patients with length gp120 protein and a canary pox vector expressing lupus, an autoimmune disease that is rarely associated the gp120/gag/protease genes reduced the risk of infec- with concurrent HIV infection, and in long-term survivors tion by 31%.8 It is unclear of HIV infection. It appears whether this is a statistical- that HIV is highly vulner- ly or clinically meaningful able to neutralization by effect. Many in the field of Studies have identified the CD4BScore specific antibodies to the HIV vaccine development CD4BScore region, but the believe that combined as the proverbial Achilles heel adaptive immune response induction of neutralizing of the virus. to the region is insufficient antibody and cytotoxic to control infection under T cells is the favored normal circumstances. A approach. As the individu- clear path to an HIV vac- al antibody and cytotoxic T cell responses to the mutable cine that induces broadly neutralizing antibodies can be HIV regions are ineffective, it is not clear how combining foreseen if the following milestones can be reached: a) these responses can be the basis for effective vaccination. Reproduction of the correct CD4BScore conformation in the Our view is that HIV vaccination will be feasible once an vaccine candidate, and b) Rapid adaptive production of immunogen is identified that induces a sufficient immune neutralizing anti-CD4BScore antibodies upon administration response to a structurally constant region of HIV essential of the vaccine candidate. for virus infection and propagation. Our preclinical studies in experimental animals based The coat protein gp41 expresses certain structural- on the covalent vaccination strategy suggest the feasibility ly conserved regions. The vaccine approach of Barton of attaining the foregoing milestones.14,15 Central points in Haynes at Duke University entails an epitope of the HIV the strategy are: gp41 coat protein located in the proximity of the lipid • The vaccine candidate, an electrophilic polypeptide membrane.9 Polyspecific antibodies that recognize this containing the CD4BScore, which binds covalently to B epitope in conjunction with membrane lipids neutralize cells, resulting in production of broadly neutralizing anti- genetically divergent HIV strains. Membranes of unin- bodies. The polypeptide is activated chemically by linking fected cells also contain the lipids as self-antigens. The lysine side chain to the strongly electrophilic phosphonate immune system is generally tolerant to the self-antigens, diester group. Naturally-occurring nucleophilic sites are and anti-HIV antibodies that react with self-antigens can found ubiquitously in B cell receptors.16,17 Noncovalent exert deleterious effects on the host. Nonetheless, there binding of the CD4BScore peptide epitope to the B cell is strong interest in the notion that breaking tolerance to receptors positions the electrophilic group within cova- self-antigens may guide development of an immunogen lent binding distance of nucleophilic groups. The ensuing capable of inducing HIV neutralizing antibodies. covalent bonding between the electrophile and nucleo- Concerning the epitopes of the CD4BS, there is no phile liberates a very large amount of energy that initiates evidence for insufficient physical exposure as the cause productive signal transduction, IgM→IgG/IgA antibody of insufficient antibody production. Similarly, an intrinsic class switching and differentiation of the cells into anti- defect in the CDR adaptive mutational process is theo- body-secreting plasma cells. retically possible, but there is no evidence that this is the • Recruitment and clonal expansion of the small subset reason for insufficient anti-CD4BS antibody production of B cells capable of producing antibodies with innate, following HIV infection or administration of the previously- pre-existing specificity directed to the CD4BScore. The

2 JUNE 2011 CD4BScore binds at a site located mainly in the framework Secretory IgA class antibodies found at mucosal surfaces regions of B cell receptors. Neutralizing antibody pro- of non-infected humans catalyze rapid gp120 cleavage duction occurs without dependence on typical adaptive and neutralize HIV in tissue culture.23 It may be hypoth- mutational processes occurring in the CDRs. However, esized that the catalytic IgAs constitute a natural defense adaptive improvement of the antibodies due to mutations against mucosal HIV transmission. of the framework regions is feasible, as suggested by In addition to inducing reversibly-binding antibodies, the evidence from immunization of animals with electrophilic covalent vaccination approach described in the preceding gp120 and an electrophilic CD4BScore peptide mimetic.18,19 section stimulates adaptive improvement of the nucleo- Robust neutralization of diverse HIV strains by the anti- philic function of antibodies. This is feasible because cova- bodies in tissue culture was evident. The antibodies dis- lent binding of the electrophilic vaccine candidate selects B played specific recognition of the CD4BScore, confirming cell receptors with the greatest nucleophilic reactivity.24,25 mimicry of the native CD4BScore by the vaccine candidates. In turn, the improved nucleophilic reactivity enhances anti- • Immunization with full-length electrophilic gp120, which body inactivation of HIV as follows. First, specific pairing overcomes the physiological hurdle in producing anti- of the antibody nucleophile with the weakly electrophilic CD4BScore antibodies. Neutralizing antibodies to elec- carbonyls of gp120 forms stable immune complexes with trophilic gp120 displayed binary epitope reactivity, that covalent character. Covalently binding antibodies were is, the simultaneous ability to bind the CD4BScore at the induced by immunization with the electrophilic analogs of antibody framework region site and a second spatially full-length gp120 and a synthetic gp120 peptide. Reversibly distant epitope at the traditional antigen binding cav- bound antibodies dissociate from HIV readily. As the cova- ity formed by the CDRs. The binary specificity suggests lent bond is very strong, the covalent antibody-HIV com- that simultaneous stimu- plexes do not dissociate, latory binding of the sec- increasing the HIV neutral- ond immunogen epitope ization potency. Second, at the CDRs compensates The neutralization potency of if the antibody combining for the down-regulatory catalytic antibodies is superior site supports water attack CD4BScore binding at the on the covalent gp120- framework regions. to traditional antibodies that bind antibody complex, catalytic the antigen reversibly on a 1:1 basis. gp120 cleavage occurs. Taken together, our stud- A subset of antibodies ies indicate the feasibil- obtained by immunization ity of developing an HIV vaccine capable of directing the with the electrophilic CD4BScore peptide catalyzed the innate CD4BS recognition capability of B cells towards a cleavage of gp120 rapidly. favorable maturational pathway, eventually resulting in Treatment of HIV using reverse transcriptase and pro- synthesis of broadly neutralizing antibodies. tease inhibitors requires vigilant management because of the potential for toxicity and emergence of drug-resistant Catalytic antibodies (Abzymes) strains. This has generated interest in passive immunother- Reversible CD4BS binding by antibodies alone is sufficient apy using monoclonal antibodies. Control of viremia upon to neutralize HIV. A subset of antibodies produced by B infusion of reversibly binding anti-HIV antibodies in humans cells express the ability to catalyze the breakdown of pep- was transient, suggesting emergence of antibody-resistant tide bonds, destroying gp120 permanently.20 A single cat- viral mutants. Very large quantities of the antibodies were alytic antibody molecule is reused to cleave thousands of necessary to reduce viral load, a reflection of modest anti- gp120 molecules over its biological half-life in blood (1–3 body neutralizing potency. Can catalytic antibodies be used weeks). The neutralization potency of catalytic antibodies, for passive immunotherapy of HIV infection? The answer therefore, is superior to traditional antibodies that bind the depends on the epitope specificity and neutralizing potency antigen reversibly on a 1:1 basis. Antibody catalytic sites of the catalysts. Targeting the CD4BScore minimizes the belong to the serine protease enzyme family, consisting opportunity for development of antibody resistant strains, as of nucleophilic sites similar to the archetypical Serine- CD4 binding and mutations in the CD4BScore are predicted Histidine-Aspartate catalytic triad of trypsin. Catalysis to result in loss of CD4 binding activity. Indeed, anti-CD4B- occurs by formation of a covalent intermediate and water Score antibodies from long-term survivors of HIV infection attack on the intermediate, regenerating an antibody mol- neutralized the autologous HIV strain potently. There is ecule that is reused for additional catalytic cycles. no evidence, therefore, for emergence of resistant strains Catalytic cleavage of gp120 occurs by noncovalent despite the selective pressure imposed by the anti-CD4B- CD4BScore binding followed by cleavage of peptide bonds. Score antibodies over prolonged durations. Anti-CD4BScore The catalytic sites are present in antibodies produced antibodies neutralize HIV in tissue culture with nanogram/ml without exposure to HIV.21,22 Sexual transmission of HIV potency, supporting their potential therapeutic application. generally occurs through the rectal and vaginal mucosal In addition to gp120, two additional HIV proteins surfaces. Only a minority of sexual intercourse events with essential for virus infection are cleaved by catalyt- an infected individual results in transmission of the virus. ic antibodies, reverse transcriptase and integrase.26,27

GMHC gmhc.org 3 is hope for translation of the preclinical immunological advances into clinical success. In the U.S., elaborate governmental arrangements are in Treatment place to prioritize the competing developmental approaches ISSUES for funding, including excellent scientific peer-review arrange- ments. However, programmatic allocation of funds is inspired Editor: Robert Valadéz Assistant Editors: Sean Cahill, Nathan Schaefer at least in part by non-scientific reasons. The literature is Art Director: Adam Fredericks replete with claims of potential clinical advances. On the GMHC Treatment Issues other hand, most HIV vaccine development projects are likely is published by GMHC, Inc. to yield incremental advances at best. An example is the All rights reserved. continued testing of vaccine formulations that induce immune Non­commercial reproduction is encouraged. responses primarily to mutable regions of HIV. Likewise, GMHC Treatment Issues intensive efforts have been undertaken to identify immune 446 W. 33 Street, New York, NY 10001 markers correlating with the marginal risk reduction observed gmhc.org in the RV144 vaccine trial. As there is doubt whether the vac- © 2011 Gay Men’s Health Crisis, Inc. cine candidate really reduced the risk of infection, it is hard to accept that meaningful correlates of risk reduction will emerge. A policy change that forthrightly admits the limited utility of classical vaccine approaches and explicitly encour- ages credible, novel approaches would be a welcome event. Scientific approaches that diverge radically from estab- Support for GMHC Treatment Issues was made possible through charitable contributions from: lished paradigms are invariably subject to rigorous peer eval- uation. Independent reproduction of the evidence is usually The Shelley & Donald Rubin Foundation necessary prior to widespread acceptance of the new scien- tific approach. These are essential safeguards against mis- Intracellular expression of catalytic antibodies to these taken conclusions and spurious claims. Antibodies obtained proteins holds potential for early blockade of viral propa- by the covalent vaccine approach have been independently gation via interference with copying viral RNA into pro- verified to neutralize diverse HIV strains in tissue culture. viral DNA and DNA integration into the host genome. Factors that might result in artifactual neutralization have Gene therapy protocols for intracellular antibody expres- been carefully eliminated.30,31 Similarly, the chemical and sion28 can be conceived for persistent delivery of cata- immunological principles underlying antibody catalysis have lytic anti-HIV antibodies. Reactivation of HIV infection been amply validated by researchers across the world. can occur due to integration of the viral genome into Occam’s razor is yet another safeguard against unproduc- host DNA. Drugs that deplete proviral DNA reservoirs tive science — when confronted with alternative explanations are under investigation to address the problem of HIV that are equal in other respects, the hypothesis that makes latency.29 Catalytic antibodies combined with a proviral the fewest novel assumptions should be selected for further DNA-depleting drug may be suitable for consideration as study. It is necessary to invoke B cell superantigenicity as an alternative therapy for the infection. the cause of poor CD4BS immunogenicity, as no compet- ing hypothesis explains the empirical findings adequately. Prioritization and funding of new technology Similarly, the innovation of covalent bonding of the vaccine development candidate to B cells is necessary, as no alternative strategy is A prophylactic vaccine and a cure for patients infected available to induce a robust anti-CD4BS antibody response. with HIV are needed urgently. However, there is con- In summary, the preclinical scientific findings support trans- siderable pessimism because of repeated clinical failure lation research aimed at realizing the clinical utility of the of candidate vaccines. The seemingly insoluble nature technology. of HIV has even inspired an argument for use of the limited available funding for improved delivery of avail- Conclusion able anti-retroviral drugs to infected patients rather than Recent immunogenicity and virus neutralization data further research investment. This argument is misguided. encourages the belief that it may be possible to develop Innovative preclinical approaches are essential if the a covalent HIV vaccine that induces broadly neutralizing objective of eradicating HIV infection is to be met. Our antibodies directed at the CD4 binding site of the virus. positive preclinical studies using the covalent vaccination Catalytic antibodies to HIV appear to be a natural defense and catalytic antibody approaches are an example. These mechanism against HIV, and it may be possible to apply approaches were developed under basic immunology broadly neutralizing catalytic antibodies as an alternative grants funded by the National Institute of Health over the therapy for HIV infection. past two decades. Additional developmental efforts will be necessary to obtain a standardized covalent vaccine and For a full list of references, author affiliations, and conflict catalytic antibody candidates for human trials, but there statement, please visit: gmhc.org/research/treatment-issues

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