Flu Universal Vaccines: New Tricks on an Old Virus

Conventional influenza vaccines are based on predicting the circulating viruses year by year, conferring limited effectiveness since the antigenicity of vaccine strains does not always match the circulating viruses. This necessitates development of universal influenza vaccines that provide broader and lasting protection against pan-influenza viruses. The discovery of the highly conserved immunogens(epitopes) of influenza viruses provides attractive targets for universal vaccine design. Here we review the current understanding with broadly protective immunogens(epitopes) and discuss several important considerations to achieve the goal of universal influenza vaccines.     

Full-Length HIV-1 Immunogens Induce Greater Magnitude and Comparable Breadth of T Lymphocyte Responses to Conserved HIV-1 Regions Compared with Conserved-Region-Only HIV-1 Immunogens in Rhesus Monkeys

A global HIV-1 vaccine will likely need to induce immune responses against conserved HIV-1 regions to contend with the profound genetic diversity of HIV-1. Here we evaluated the capacity of immunogens consisting of only highly conserved HIV-1 sequences that are aimed at focusing cellular immune responses on these potentially critical regions. We assessed in rhesus monkeys the breadth and magnitude of T lymphocyte responses elicited by adenovirus vectors expressing either full-length HIV-1 Gag/Pol/Env immunogens or concatenated immunogens consisting of only highly conserved HIV-1 sequences. Surprisingly, we found that the full-length immunogens induced comparable breadth (P = 1.0) and greater magnitude (P = 0.01) of CD8⁺ T lymphocyte responses against conserved HIV-1 regions compared with the conserved-region-only immunogens. Moreover, the full-length immunogens induced a 5-fold increased total breadth of HIV-1-specific T lymphocyte responses compared with the conserved-region-only immunogens (P = 0.007). These results suggest that full-length HIV-1 immunogens elicit a substantially increased magnitude and breadth of cellular immune responses compared with conserved-region-only HIV-1 immunogens, including greater magnitude and comparable breadth of responses against conserved sequences.     

The first hypervariable region of the gp120 Env glycoprotein defines the neutralizing susceptibility of heterologous human immunodeficiency virus type 1 isolates to neutralizing antibodies elicited by the SF162gp140 immunogen

Current vaccine efforts to elicit cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus (HIV) focus on the engineering of soluble mimetics of the trimeric HIV Env glycoprotein (commonly termed gp140 immunogens). Such immunogens are thought to be more effective than previously tested monomeric gp120 immunogens at eliciting cross-reactive NAbs. Still, the breadth of neutralizing antibody responses elicited by gp140 immunogens is narrow. Understanding why antibodies elicited by gp140 immunogens fail to neutralize a wide range of heterologous primary HIV isolates is necessary for improving the design of such immunogens. We previously reported that antibodies elicited in macaques by SF162 Env-derived gp140 immunogens fail to neutralize several heterologous “neutralization-resistant” primary HIV type 1 isolates, such as JRFL, ADA, and YU2. Here we show that by replacing the V1 region of Env on these heterologous viruses with that of SF162, we render them highly susceptible to neutralization by the SF162gp140-elicited antibodies. We observed that viral neutralization was mediated not only by vaccine-elicited anti-V1 but also by anti-V3 antibodies and antibodies directed against as yet unidentified Env regions, depending on the heterologous Env background. Our study indicates that common neutralization epitopes are differentially exposed on diverse primary HIV isolates and that the V1 loop contributes to this differential exposure. Therefore, the antibody responses elicited by soluble gp140 immunogens will have to overcome several distinct obstacles in order to neutralize diverse primary HIV isolates.     

Protective Efficacy of Centralized and Polyvalent Envelope Immunogens in an Attenuated Equine Lentivirus Vaccine

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.     

Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.     

Vehicles for genetic vaccines against human immunodeficiency virus: induction of T cell-mediated immune responses

Success of a candidate vaccine against human immunodeficiency virus (HIV) depends on the type, site, strength, longevity and specificity of the immune responses it induces. The specificity of a vaccine is determined by the HIV-derived immunogens it employs in its formulation. Central to the other features is a correct and efficient delivery of the immunogens to the relevant cells of the immune system, which leads to orchestrated actions of millions of cells of several types and functions at multiple sites in the body. Thus, for elicitation of cytotoxic T lymphocyte responses, immunogens have to be delivered to the so called 'professional' antigen-presenting cells in a way that leads to a specific activation and expansion of na?ve or precursor T cells, subsequent maturation of effector functions and, importantly, generation of a potent immunological memory. Many aspects of theseprocesses are currently unknown. However, it is very likely that the immunogenicities of genetic vaccines, i.e. vaccines delivering genes coding for immunogens rather than purified possibly adjuvanted proteins or peptides themselves, are in great part determined by the choice of vaccine vehicles and route of administration. In addition, vaccine immunogenicities can be augmented semi-rationally by immunogen engineering and co-delivering immunomodulatory molecules, and empirically by combining different vehicles expressing the same immunogen in heterologous prime-boost protocols. In any case, a successful vaccination strategy against HIV as well as other chronic viral infections has to elicit better immune responses than the natural infections do.     

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.     

Cross-subtype T-cell immune responses induced by a human immunodeficiency virus type 1 group m consensus env immunogen

The genetic diversity among globally circulating human immunodeficiency virus type 1 (HIV-1) strains is a serious challenge for HIV-1 vaccine design. We have generated a synthetic group M consensus env gene (CON6) for induction of cross-subtype immune responses and report here a comparative study of T-cell responses to this and natural strain env immunogens in a murine model. Three different strains of mice were immunized with CON6 as well as subtype A, B, or C env immunogens, using a DNA prime-recombinant vaccinia virus boost strategy. T-cell epitopes were mapped by gamma interferon enzyme-linked immunospot analysis using five overlapping Env peptide sets from heterologous subtype A, B, and C viruses. The CON6-derived vaccine was immunogenic and induced a greater number of T-cell epitope responses than any single wild-type subtype A, B, and C env immunogen and similar T-cell responses to a polyvalent vaccine. The responses were comparable to within-clade responses but significantly more than between-clade responses. The magnitude of the T-cell responses induced by CON6 (measured by individual epitope peptides) was also greater than the magnitude of responses induced by individual wild-type env immunogens. Though the limited major histocompatibility complex repertoire in inbred mice does not necessarily predict responses in nonhuman primates and humans, these results suggest that synthetic centralized env immunogens represent a promising approach for HIV-1 vaccine design that merits further characterization.     

Coping with viral diversity in HIV vaccine design

The ability of human immunodeficiency virus type 1 (HIV-1) to develop high levels of genetic diversity, and thereby acquire mutations to escape immune pressures, contributes to the difficulties in producing a vaccine. Possibly no single HIV-1 sequence can induce sufficiently broad immunity to protect against a wide variety of infectious strains, or block mutational escape pathways available to the virus after infection. The authors describe the generation of HIV-1 immunogens that minimizes the phylogenetic distance of viral strains throughout the known viral population (the center of tree [COT]) and then extend the COT immunogen by addition of a composite sequence that includes high-frequency variable sites preserved in their native contexts. The resulting COT⁺ antigens compress the variation found in many independent HIV-1 isolates into lengths suitable for vaccine immunogens. It is possible to capture 62% of the variation found in the Nef protein and 82% of the variation in the Gag protein into immunogens of three gene lengths. The authors put forward immunogen designs that maximize representation of the diverse antigenic features present in a spectrum of HIV-1 strains. These immunogens should elicit immune responses against high-frequency viral strains as well as against most mutant forms of the virus.     

Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.     

Human Rhinovirus Type 14:Human Immunodeficiency Virus Type 1 (HIV-1) V3 Loop Chimeras from a Combinatorial Library Induce Potent Neutralizing Antibody Responses against HIV-1

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.     

Soluble HIV-1 Envelope Immunogens Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low Potency Neutralizing Antibodies

We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses.     

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

Limitations to the structure-based design of HIV-1 vaccine immunogens

In spite of 25 years of intensive research, no effective human immunodeficiency virus type 1 (HIV-1) vaccine has yet been developed. One reason for this is that investigators have concentrated mainly on the structural analysis of HIV-1 antigens because they assumed that it should be possible to deduce vaccine-relevant immunogens from the structure of viral antigens bound to neutralizing monoclonal antibodies. This unwarranted assumption arises from misconceptions regarding the nature of protein epitopes and from the belief that it is justified to extrapolate from the antigenicity to the immunogenicity of proteins. Although the structure of the major HIV-1 antigenic sites has been elucidated, this knowledge has been of little use for designing an HIV-1 vaccine. Little attention has been given to the fact that protective immune responses tend to be polyclonal and involve antibodies directed to several different epitopes. It is concluded that only trial and error, empirical investigations using numerous immunization protocols may eventually allow us to identify which mixtures of immunogens are likely to be the best candidates for an HIV-1 vaccine.     

Identification of Non-HIV Immunogens That Bind to Germline b12 Predecessors and Prime for Elicitation of Cross-clade Neutralizing HIV-1 Antibodies

A fundamental challenge for developing an effective and safe HIV-1 vaccine is to identify vaccine immunogens that can initiate and maintain immune responses leading to elicitation of broadly neutralizing HIV-1 antibodies (bnAbs) through complex maturation pathways. We have previously found that HIV-1 envelope glycoproteins (Env) lack measurable binding to putative germline predecessors of known bnAbs and proposed to search for non-HIV immunogens that could initiate their somatic maturation. Using bnAb b12 as a model bnAb and yeast display technology, we isolated five (poly)peptides from plant leaves, insects, E. coli strains, and sea water microbes that bind to b12 putative germline and intermediate antibodies. Rabbit immunization with the (poly)peptides alone induced high titers of cross-reactive antibodies that neutralized HIV-1 isolates SF162 and JRFL. Priming rabbits with the (poly)peptides followed by boosts with trimeric gp140_SF162 and then resurfaced Env (RSC3) induced antibodies that competed with mature b12 and neutralized tier 1 and 2 viruses from clade B, C and E, while control rabbits without (poly)peptide priming induced antibodies that did not compete with mature b12 and neutralized fewer isolates. The degree of competition with mature b12 for binding to gp140_SF162 correlated with the neutralizing activity of the rabbit IgG. Reversing the order of the two boosting immunogens significantly affected the binding profile and neutralization potency of the rabbit IgG. Our study is the first to provide evidence that appears to support the concept that non-HIV immunogens may initiate immune responses leading to elicitation of cross-clade neutralizing antibodies.     

Identification of Brugia malayi immunogens by an immunoproteomics approach

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis.     

Progress in the rational design of an AIDS vaccine

Human immunodeficiency virus-1 (HIV-1) has a high degree of genetic and antigenic diversity that has impeded the development of an effective vaccine using traditional methods. We are attempting to develop an AIDS vaccine by employing strategies that include structural biology and computational modelling, in an effort to develop immunogens capable of eliciting neutralizing antibodies of the requisite breadth and potency against circulating strains of HIV-1.     

Cross-clade HIV-1 neutralizing antibodies induced with V3-scaffold protein immunogens following priming with gp120 DNA

The V3 epitope is a known target for HIV-1 neutralizing antibodies (NAbs), and V3-scaffold fusion proteins used as boosting immunogens after gp120 DNA priming were previously shown to induce NAbs in rabbits. Here, we evaluated whether the breadth and potency of the NAb response could be improved when boosted with rationally designed V3-scaffold immunogens. Rabbits were primed with codon-optimized clade C gp120 DNA and boosted with one of five V3-cholera toxin B fusion proteins (V3-CTBs) or with double combinations of these. The inserts in these immunogens were designed to display V3 epitopes shared by the majority of global HIV-1 isolates. Double combinations of V3-CTB immunogens generally induced more broad and potent NAbs than did boosts with single V3-CTB immunogens, with the most potent and broad NAbs elicited with the V3-CTB carrying the consensus V3 of clade C (V3(C)-CTB), or with double combinations of V3-CTB immunogens that included V3(C)-CTB. Neutralization of tier 1 and 2 pseudoviruses from clades AG, B, and C and of peripheral blood mononuclear cell (PBMC)-grown primary viruses from clades A, AG, and B was achieved, demonstrating that priming with gp120 DNA followed by boosts with V3-scaffold immunogens effectively elicits cross-clade NAbs. Focusing on the V3 region is a first step in designing a vaccine targeting protective epitopes, a strategy with potential advantages over the use of Env, a molecule that evolved to protect the virus by poorly inducing NAbs and by shielding the epitopes that are most critical for infectivity.     

Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.     

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Introduction: Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens.

Areas covered: We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike.

Expert commentary: Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates.