HIV Vaccines: One Step Closer

Spotlight

HIV Vaccines: One Step Closer Michael Sze Yuan Low1,2,3,4 and David Tarlinton4,* Currently there is no effective vaccine against human immunodeficiency virus (HIV). Four recently published studies in Cell and Immunity now show that using planned sequential boosting with antigens to guide the humoral response towards broadly neutralizing antibodies could provide a solution to achieving vaccination against HIV-1. Despite improvements in prevention and therapies against HIV and its clinical consequence, AIDS, over 36 million people are estimated to be infected worldwide [1]. Highly active anti-retroviral therapeutic agents (HAART) can reduce patients’ viral load, reducing the risk of AIDS and transmission to contacts. However, HAART cannot currently eradicate HIV owing to the known reservoir of infected cells thought to predominantly consist of CD4+ memory T cells [2]. With newer findings, broadly neutralizing antibodies (bnAbs) appear to hold the promise of both long-term drug-free disease control in infected patients, as well as effective community-based prevention via vaccination. BnAbs occur in natural HIV infection, but typically only develop after years of infection. Importantly, the genes encoding their immunoglobulin variable (IgV) regions contain remarkably high amounts of somatic hypermutation [3]. In addition, they all harbor unusual antigen binding sites, with particularly long and short CDR3 loops in their immunoglobulin heavy and light chains, respectively [3]. BnAbs target invariant and crucial molecules on the virus, neutralizing the ability of HIV to infect cells and replicate. Several bnAbs have

been described and have been recently reviewed [3]. In general terms, HIV bnAbs recognize epitopes on the viral envelope, with the CD4 binding site being a common, but not universal, target [3]. There are several reasons why the induction of bnAbs against HIV using vaccination has thus far been unsuccessful. First, HIV is an extremely diverse virus, with multiple subtypes leading to antigenic variation of up to 30% within its viral envelope [4,5]. Second, the envelope is comprised of numerous glycans that shield antigenic sites and can undergo conformational change, leading to a rapidly transforming antigenic landscape [3,4]. Lastly, bnAbs lose their ability to neutralize HIV when their immunoglobulin variable (V) gene sequences are reverted to their unmutated (germline) counterparts [3,4]. This raises the quandary of how unmutated, naïve B cells that potentially encode bnAbs can be induced to participate in an immune response, and then be guided through rounds of somatic hypermutation and selection to produce the specificity and affinity of a given bnAb. This last point is particularly pertinent as current vaccination techniques rely on exposure to a set of pathogen-specific antigens, with subsequent boosts that involve restimulation with the same antigens. A major exception to this approach is the influenza vaccine in which antigens are altered each year to target new seasonal strains arising from antigenic drift. Influenza vaccines, however, are not specifically designed to restimulate existing memory B cells into further affinity maturation, but are likely to activate naïve B cells. Currently, no mainstream vaccinations alter antigenic stimulation in a systematic and staged manner to guide the antibody response towards a desired epitope specificity. In contrast to the existing paradigm, a staged immunization program appears to be the current best hope for developing an effective vaccination strategy based on eliciting bnAbs against HIV. Recently, four different, concurrent publications showed

that sequential immunization could elicit bnAbs against two important HIV-1 envelope targets [6–9]. The first bnAb investigated was PGT-121, an antibody that targets the third variable loop (V3) on the viral envelope. Using a ‘PGT-121 knock-in’ mouse, that carries the inferred germline heavy and light chains of the PGT-121 antibody, the authors showed that, without immunization, mice lacked bnAbs [7,8]. Importantly, by predicting the changes that occur with recurrent somatic hypermutation, the authors were able to ‘shepherd’ the antibody affinity towards that of the PGT-121 bnAb. This was achieved by altering antigenic stimulation using trimers of different conformations [7,8]. This specific and sequential immunization scheme thus allowed the production of bnAbs that were highly similar to the original PGT-121 bnAb. Two other groups produced similar results by inducing VRC01-like bnAbs that primarily recognize the CD4 binding site on the HIV-1 envelope. A previous study had shown that an immunogen, eOD-GT8 60mer, could stimulate naïve B cells down the path towards VRC01 bnAbs in a mouse model termed VRC01 gH in which the majority of B cells expressed the germline heavy chain of the VRC01 antibody [10]. Importantly, the eOD-GT8 60mer alone could not induce a complete bnAb-like response. Instead, much like with PGT-121 antibodies, a systematic and sequential method of antigenic stimulation led to the production of VRC01-like bnAbs, guiding the immune response and somatic hypermutation towards the specific characteristics of the original VRC01 bnAb [6,9]. There are two important similarities between these published studies. First, the authors have overcome a major limitation of research into HIV by demonstrating mouse models for screening vaccination strategies that lead to bnAbs [6–9]. Establishing such models allows

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research to be performed at a higher efficiency than in larger animal models such as macaques. Second, all studies used a staged and highly-deliberate boosting

strategy comprising 4–6 boosts to guide antibody specificity away from germline, and towards the unusual and highly mutated bnAbs (Figure 1). Furthermore,

Germinal center

the fact that sequential immunization has been successfully performed for two structurally different bnAbs suggests that this methodology could be used in other diseases in which standard vaccination techniques have failed.

Anbody-secreng cell with affinity to new angen

The success seen in these studies suggests that sequential immunization for eliciting HIV-1 bnAbs is a strategy worthy of further investigation. Nonetheless, looking into the future, several important questions remain unanswered. First, which bnAb should be used for a more in-depth Mutated memory investigation, with the ultimate aim of translating this vaccination approach to B cell humans? There are over 32 known bnAbs and choosing which one(s) to elicit with Anbody-secreng vaccinations will be important for future cell with new affinity to work [3]. Second, how effective would a new angen sequential vaccination program be if one or more doses of the vaccination regimen were to be missed? Rates of missed vaccinations vary between countries but are relatively common. Given the strategic and carefully planned nature of the boosting Mutated memory regimens involved in these studies, future B cell work would also need to consider the efficacy of the resulting antibodies if some of the boosts were not administered. Third, all of the mice were enriched significantly for the specificity of the relevant germline-encoded bnAbs. Determining Mutated memory to what extent this enlarged precursor B cell frequency influences the vaccination outcome will also be crucial. Regardless, collectively, these recent and exciting findings suggest that a future where effective HIV vaccination regimens are accessible may not lie too far ahead.

Angen

B cell

Altered angen

Mutated memory B cell

Altered angen

Mutated memory B cell

Anbody-secreng cell with new affinity to new angen

Acknowledgments M.L. is funded by a C.R.B. Blackburn scholarship jointly from the National Health and Medical Research

Anbodies with Broadly neutralizing Capabilies

Council (NHMRC) Australia and Royal Australasian College of Physicians, and D.T. by a NHMRC research fellowship. Work in the authors’ laboratory is supported by an NHMRC program grant (1054925).

Figure 1. Development of Broadly Neutralizing Antibodies (bnAbs) with Desired Epitope Specificity. A schematic is shown of how planned, sequential boosting using different antigens can allow the development of antibodies with desired epitope specificity. This process is undertaken 4–6 times to obtain broadly neutralizing anti-HIV-1 antibodies.

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1 Immunology Division, Walter and Eliza Hall Institute of Medical Research, University of Melbourne, 1G Royal

Parade, Parkville, Victoria, Australia 2 Department of Haematology, Monash Health, Monash Hospital, 246 Clayton Road Clayton, Victoria, Australia

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Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia 4 Department of Immunology and Pathology, Monash University, Melbourne, Victoria 3004, Australia *Correspondence: [email protected] (D. Tarlinton). http://dx.doi.org/10.1016/j.molmed.2016.10.006 References 1. Joint United Nations Programme on HIV/AIDS (UNAIDS) (2016) Global AIDS Update, World Health Organisation 2. Ruelas, D.S. and Greene, W.C. (2013) An integrated overview of HIV-1 latency. Cell 155, 519–529 3. Wu, X. and Kong, X.P. (2016) Antigenic landscape of the HIV-1 envelope and new immunological concepts defined by HIV-1 broadly neutralizing antibodies. Curr. Opin. Immunol. 42, 56–64 4. Klein, F. et al. (2013) Antibodies in HIV-1 vaccine development and therapy. Science 341, 1199–1204 5. Gaschen, B. et al. (2002) Diversity considerations in HIV-1 vaccine selection. Science 296, 2354–2360 6. Briney, B. et al. (2016) Tailored immunogens direct affinity maturation toward HIV neutralizing antibodies. Cell 166, 1459–1470 7. Escolano, A. et al. (2016) Sequential immunization elicits broadly neutralizing anti-HIV-1 antibodies in Ig knockin mice. Cell 166, 1445–1458 8. Steichen, J.M. et al. (2016) HIV vaccine design to target germline precursors of glycan-dependent broadly neutralizing antibodies. Immunity 45, 483–496 9. Tian, M. et al. (2016) Induction of HIV neutralizing antibody lineages in mice with diverse precursor repertoires. Cell 166, 1471–1484 10. Jardine, J.G. et al. (2015) Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen. Science 349, 156–161

Spotlight

Fumarates and Cancer Gwenny M. Fuhler,1,* Hester Eppinga,1 and Maikel P. Peppelenbosch1 Accumulation of intermediate metabolites of the tricarboxylic acid (TCA) cycle in tumor cells can cause epithelial-to-mesenchymal transition (EMT), although the exact mechanisms remain elusive. Recent studies show that the oncometabolite fumarate, which accumulates in fumarate hydratase-deficient renal cancers, confers tumor aggressiveness by causing epigenetic changes in

the antimetastatic miRNA cluster mir-200ba429. This may have important implications for the use of fumarates in the clinic. In addition to oncogenic driver mutations, many tumors acquire alterations in cellular metabolism. Generation of energy in normal, differentiated cells starts with glycolysis, where glucose is metabolized to pyruvate, producing two ATP molecules. In the presence of oxygen, this is followed by conversion of pyruvate to acetyl coenzyme A, allowing initiation of the TCA cycle and further energy harvest in the mitochondrial electron transport chain. This whole process yields a total of up to 38 ATP molecules. Under hypoxic conditions, pyruvate is fermented to lactate in a process called ‘anaerobic glycolysis’, with a yield of only two ATP molecules per glucose molecule. However, in cancer cells, most glucose is converted to lactate, even in the presence of oxygen, which is known as the ‘Warburg effect’, or ‘aerobic glycolysis’. The reason why tumor cells would use such an inefficient energy conversion pathway is much speculated upon and, as yet, incompletely understood. Although this effect is not thought to be related to mitochondrial respiration defects, it is noteworthy that several tumors show mutations in genes regulating the TCA cycle, thereby increasing its intermediate metabolites. Some of these metabolites have been shown to contribute to oncogenic processes and have been termed ‘oncometabolites’ [1]. Thus, it is tempting to speculate that the presence of aerobic glycolysis is permissive for the acquisition of oncogenic mutations in the TCA cycle in some tumors, which would otherwise have been detrimental to cell survival. The best-described TCA enzyme mutations to date are those in the genes encoding isocitrate dehydrogenase (IDH1/IDH2), succinate dehydrogenase (SDH), and fumarate hydratase (FH) (Figure 1). While the mutations in SDH and FH are inactivating and result in the accumulation of

succinate and fumarate, respectively, gain-of-function mutations in IDH cause the accumulation of D-2-hydroxyglutarate (2-HG), a reduced form of /-ketoglutarate, normally kept at low levels in the cell. It was previously demonstrated that succinate and 2-HG induce EMT in ovarian cancer and colorectal cancer (CRC) cells, respectively [2,3]. The latter study further showed that 2-HG-induced changes in EMT-associated gene expression (e.g., increased Vimentin and decreased E-cadherin levels) were dependent on increased expression of the transcription factors ZEB1 and ZEB2 in human epithelial cell lines, whereas other known EMT drivers, such as ZEB2, SNAI1 (Snail), and SNAI2 (Slug), were not required [3]. ZEB1, in turn, is regulated by the antimetastatic miRNA-200 family, which was shown to be downregulated in IDH1mutated epithelial cells [3]. This limited knowledge of oncometabolite function has now been extended through a recent article by Sciacovelli et al. published in Nature [4]. The authors demonstrated that increased cellular fumarate levels in fumarate hydratase (Fh1)-deficient murine renal tumors also drive EMT changes, including the upregulation of Vimentin, Snai2, Zeb1, and Zeb2; and provided an in-depth analysis of some of the underlying molecular mechanisms of EMT in this context (Figure 1). Sciacovelli et al. showed that EMT was dependent on the Ten-Eleven Translocation proteins (Tets) 2 and 3. These methylcytosine dioxygenases convert 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC), the levels of which were decreased in Fh1deficient cells, thus suggesting that fumarate directly inhibits Tet activity. 5-hmC is an oxidative intermediate in the cytosine demethylation pathway produced when Tets bind DNA, and the authors went on to demonstrate that CpG43, a conserved GpG island at the 50 end of mir200ba429, is a target of Tets. Tet inactivation in Fh1-deficient cells resulted in CpG43 hypermethylation, consistent with reduced expression of miRNA-200a and miR-200b family members upon

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