Recombinant Env Protein Research Articles

Characterization and application of recombinant Bovine Leukemia Virus Env protein

The Bovine Leukemia Virus (BLV) Envelope (Env) glycoprotein complex is instrumental in viral infectivity and shapes the host’s immune response. This study presents the production and characterization of a soluble furin-mutated BLV Env ectodomain (sBLV-EnvFm) expressed in a stable S2 insect cell line. We purified a 63 kDa soluble protein, corresponding to the monomeric sBLV-EnvFm, which predominantly presented oligomannose and paucimannose N-glycans, with a high content of core fucose structures. Our results demonstrate that our recombinant protein can be recognized from specific antibodies in BLV infected cattle, suggesting its potential as a powerful diagnostic tool. Moreover, the robust humoral immune response it elicited in mice shows its potential contribution to the development of subunit-based vaccines against BLV.

Human Endogenous Retrovirus K Envelope in Spinal Fluid of Amyotrophic Lateral Sclerosis Is Toxic.

Human endogenous retroviruses have been implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Expression of human endogenous retrovirus K (HERV-K) subtype HML-2 envelope (Env) in human neuronal cultures and in transgenic mice results in neurotoxicity and neurodegeneration, and mice expressing HML-2 Env display behavioral and neuromuscular characteristics resembling ALS. This study aims to characterize the neurotoxic properties of HML-2 Env. Env neurotoxicity was detected in ALS cerebrospinal fluid and confirmed using recombinant Env protein in a cell-based assay and a mouse model. The mechanism of neurotoxicity was assessed with immunoprecipitation followed by mass spectrometry and Western blot, and by screening a panel of inhibitors. We observed that recombinant HML-2 Env protein caused neurotoxicity resulting in neuronal cell death, retraction of neurites, and decreased neuronal electrical activity. Injection of the Env protein into the brains of mice also resulted in neuronal cell death. HML-2 Env protein was also found in the cerebrospinal fluid of patients with sporadic ALS. The neurotoxic properties of the Env and the cerebrospinal fluid could be rescued with the anti-Env antibody. The Env was found to bind to CD98HC complexed to β1 integrin on the neuronal cell surface. Using a panel of compounds to screen for their ability to block Env-induced neurotoxicity, we found that several compounds were protective and are linked to the β1 integrin pathway. HERV-K Env is released extracellularly in ALS and causes neurotoxicity via a novel mechanism. Present results pave the way for new treatment strategies in sporadic ALS. ANN NEUROL 2022;92:545-561.

Post-Immune Antibodies in HIV-1 Infection in the Context of Vaccine Development: A Variety of Biological Functions and Catalytic Activities.

Unlike many other viruses, HIV-1 is highly variable. The structure of the viral envelope changes as the infection progresses and is one of the biggest obstacles in developing an HIV-1 vaccine. HIV-1 infection can cause the production of various natural autoantibodies, including catalytic antibodies hydrolyzing DNA, myelin basic protein, histones, HIV-integrase, HIV-reverse transcriptase, β-casein, serum albumin, and some other natural substrates. Currently, there are various directions for the development of HIV-1 vaccines: stimulation of the immune response on the mucous membranes; induction of cytotoxic T cells, which lyse infected cells and hold back HIV-infection; immunization with recombinant Env proteins or vectors encoding Env; mRNA-based vaccines and some others. However, despite many attempts to develop an HIV-1 vaccine, none have been successful. Here we review the entire spectrum of antibodies found in HIV-infected patients, including neutralizing antibodies specific to various viral epitopes, as well as antibodies formed against various autoantigens, catalytic antibodies against autoantigens, and some viral proteins. We consider various promising targets for developing a vaccine that will not produce unwanted antibodies in vaccinated patients. In addition, we review common problems in the development of a vaccine against HIV-1.

Autoantibodies against the envelope proteins of endogenous retroviruses K102 and K108 in patients with systemic lupus erythematosus correlate with active disease.

To determine if patients with systemic lupus erythematosus (SLE), a disease characterised by elevated type I interferons reminiscent of anti-viral immunity, have expression of human endogenous retrovirus K (HERV-K) proviruses capable of producing envelope (Env) protein, as well as associated autoantibodies against the Env protein. ELISAs were conducted with recombinant Env protein and sera from SLE patients with active (n=60) or inactive (n=49) disease, healthy controls (n=47), other rheumatic disorders (n=59), as well as plasma from paediatric lupus patients with active (n=30) or inactive (n=30) disease, and 17 healthy children. Antibody reactivity was evaluated for correlations with clinical and laboratory parameters of the patients. Expression of HERV-K transcripts were profiled in SLE leukocytes by RNA-Seq. Both adult and paediatric SLE patients had autoantibodies against HERV-K Env with higher titres than healthy controls or patients with Sjögren's syndrome, small- or large-vessel vasculitis, or psoriatic arthritis. Transcripts from only two HERV-K loci capable of producing Env, HERV-K102 and -K108, were detected among the 10 expressed loci in SLE patients. Our data reveal that HERV-K proviruses are expressed in SLE and that the HERV-K-encoded Env protein elicits an immune response in patients, particularly during active disease.

Therapeutic vaccination of koalas harbouring endogenous koala retrovirus (KoRV) improves antibody responses and reduces circulating viral load

The long-term survival of the koala is under serious threat from multiple factors, including infectious disease agents such as Chlamydia and koala retrovirus (KoRV). KoRV is present in both exogenous and endogenous forms, depending on the geographical location of the population. In the northern half of Australia, it is present as an endogenous infection in all koalas, making a case for an urgent need to develop a therapeutic vaccine that might prevent KoRV-associated pathologies in these koalas. To this end, we determined the therapeutic effects of vaccinating koalas harbouring endogenous KoRV with a recombinant KoRV Env protein combined with a Tri-adjuvant. We found that vaccination led to a significant increase in circulating anti-KoRV IgG levels, as well as increase in neutralising antibodies. Our study also showed that post-vaccination antibodies were able to recognize epitopes on the Env protein that were unrecognised pre-vaccination, as well as resulting in an increase in the recognition of the previously recognised epitopes. The vaccine also induced antibodies that were cross-reactive against multiple KoRV-subtypes. Finally, we found a complete clearance of KoRV-A in plasma from koalas that had detectable levels of KoRV-A pre-vaccination. Similarly, there was a significant reduction in the expression of KoRV-B viral RNA levels post-vaccination. Collectively, this study showed that koalas harbouring endogenous KoRV can benefit from prophylactic vaccination against KoRV using a recombinant KoRV-A Env protein and that the mechanism of this protection might be through the boosting of natural anti-KoRV antibodies and expanding the breadth of the recognised epitopes.

Gene editing in CHO cells to prevent proteolysis and enhance glycosylation: Production of HIV envelope proteins as vaccine immunogens.

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of -recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/- CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/- MGAT1- CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the N-linked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/- MGAT1- CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions.

Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components.IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

Integrity of Glycosylation Processing of a Glycan-Depleted Trimeric HIV-1 Immunogen Targeting Key B-Cell Lineages.

Broadly neutralizing antibodies (bNAbs) that target the trimeric HIV-1 envelope glycoprotein spike (Env) are tools that can guide the design of recombinant Env proteins intended to engage the predicted human germline precursors of bNAbs (gl-bNAbs). The protein components of gl-bNAb epitopes are often masked by glycans, while mature bNAbs can evolve to accommodate or bypass these shielding glycans. The design of germline-targeting Env immunogens therefore includes the targeted deletion of specific glycan sites. However, the processing of glycans on Env trimers can be influenced by the density with which they are packed together, a highly relevant point given the essential contributions under-processed glycans make to multiple bNAb epitopes. We sought to determine the impact of the removal of 15 potential N-glycan sites (5 per protomer) from the germline-targeting soluble trimer, BG505 SOSIP.v4.1-GT1, using quantitative, site-specific N-glycan mass spectrometry analysis. We find that, compared with SOSIP.664, there was little overall change in the glycan profile but only subtle increases in the extent of processing at sites immediately adjacent to where glycans had been deleted. We conclude that multiple glycans can be deleted from BG505 SOSIP trimers without perturbing the overall integrity of the glycan shield.

Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques.

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research.

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and Tcells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

Germline-targeting immunogens.

In 2009, Dimitrov's group reported that the inferred germline (iGL) forms of several HIV-1 broadly neutralizing antibodies (bNAbs) did not display measurable binding to a recombinant gp140 Env protein (derived from the dual-tropic 89.6 virus), which was efficiently recognized by the mature (somatically mutated) antibodies. At that time, a small number of bNAbs were available, but in the following years, the implementation of high-throughput B-cell isolation and sequencing assays and of screening methodologies facilitated the isolation of greater numbers of bNAbs from infected subjects. Using these newest bNAbs, and a wide range of diverse recombinant Envs, we and others confirmed the observations made by Dimitrov's group. The results from these studies created a paradigm shift in our collective thinking as to why recombinant Envs are ineffective in eliciting bNAbs and has led to the "germline-targeting" immunization approach. Here we discuss this approach in detail: what has been done so far, the advantages and limitations of the current germline-targeting immunogens and of the animal models used to test them, and we conclude with a few thoughts about future directions in this area of research.

Detection of HIV Env-mediated B cell receptor aggregation on engineered B cells by flow cytometry.

Abstract Approximately 20% of individuals infected with HIV-1 produce broadly neutralizing antibodies (bNAbs) against the virus. Understanding how bNAb linked B cell receptors (BCRs) are initially stimulated by the HIV-1 envelope (Env) in the context of natural HIV infection is a major focus of HIV research. While germline-reverted BCR forms of bNAbs do not efficiently recognize Env, recombinant Env proteins have been recently designed to engage and activate some germline BCRs. To better understand how such modified Envs engage BCRs and to further optimize the activation of B cells, we (a) created a stable Ramos cell line that expresses the germline BCR form of a potent and broadly neutralizing antibody, VRC01 and (b) developed a fluorescence-based assay that allows for the monitoring of Env-mediated BCR aggregation. B cells were incubated with different multimeric forms of Env (valency ranging from 1–24) and determined the extent of BCR aggregation and compared it with the extent of B cell activation (measured by Ca2+flux assays). Significant increases in BCR aggregation and B cell-activation were detected in response to heptameric and 24-meric forms of Env. In contrast, lower valency multimeric forms of Env activated B cells inefficiently. No aggregation or calcium flux was detected in response to monomeric Env. A positive correlation was observed between the magnitudes of calcium flux and BCR aggregation. Thus, either BCR aggregation or Ca2+ flux assays can be used to monitor the activation of B cells by recombinant Env proteins that are being evaluated as possible immunogens to elicit bNAbs. Our results are relevant to optimizing the design and formulation of recombinant Env proteins for the elicitation of broadly neutralizing HIV-1 antibodies.

Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins.

We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505 env gene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds. It is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

Multimodality vaccination against clade C SHIV: Partial protection against mucosal challenges with a heterologous tier 2 virus

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses.

Vaccine-induced HIV-1 envelope gp120 constant region 1-specific antibodies expose a CD4-inducible epitope and block the interaction of HIV-1 gp140 with galactosylceramide.

Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site. Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.

Dysfunction of Bovine Endogenous Retrovirus K2 Envelope Glycoprotein Is Related to Unsuccessful Intracellular Trafficking

Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry receptor and virus-host membrane fusion, respectively. However, unexplained issues remain for the maturation process of retroviral Env. We previously reported that bovine endogenous retrovirus K2 (BERV-K2) Env lost fusogenicity due to a defect in the cleavage of SU and TM. In this study, we identified that mutations residing in BERV-K2 SU disturbed intracellular trafficking of BERV-K2 Env and resulted its inefficient cleavage. Because SU is not known to play an important role in this process, our study may provide novel insights into the maturation mechanism of retroviral Envs.

HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

Utility of the Sindbis replicon system as an Env-targeted HIV vaccine

Sindbis replicon-based vaccine vectors are designed to combine the immunostimulatory properties of replicating viruses with the superior safety profile of non-replicating systems. In this study we performed a detailed assessment of Sindbis (SIN) replicon vectors expressing HIV-1 envelope protein (Env) for the induction of cell-mediated and humoral immune responses in a small animal model. SIN-derived virus-like particles (VLP) elicited Env-specific antibody responses that were detectable after boosting with recombinant Env protein. This priming effect could be mediated by replicon activity alone but may be enhanced by Env attached to the surface of VLP, offering a potential advantage for this mode of replicon delivery for Env based vaccination strategies. In contrast, the Env-specific CTL responses that were elicited by SIN-VLP were entirely dependent on replicon activity. SIN-VLP priming induced more durable humoral responses than immunization with protein only. This is important from a vaccine perspective, given the intrinsic tendency of Env to induce short-lived antibody responses in the context of vaccination or infection. These results indicate that further efforts to enhance the magnitude and durability of the HIV-1 Env-specific immune responses generated by Sindbis vectors, either alone or as part of prime-boost regimens, are justified.

Recombinant Env proteins that bind the quaternary-specific, V1/V2-directed PGT antibodies

Background Antibodies PGT141-145 are broadly neutralizing and recognize a glycan-dependent epitope in the V1/V2 loop, similar to antibodies PG9 and PG16. Collectively, this class of antibodies binds preferentially to the functional viral spike. Although PG9, and to a lesser extent, PG16, bind monomeric gp120s and V1/V2 scaffolds, to date no recombinant env-derived proteins have been identified that bind to antibodies PGT141-145.

Co-immunization with HIV Env DNA and protein elicit long-lasting strong cellular and humoral immune responses

Background We have previously reported that potent, long-lasting HIV-1 Env-specific cell-mediated immune responses could be elicited in rhesus macaques and mice using plasmids encoding env DNA as the immunogen. Subsequent experiments showed that combination of DNA and protein in the form of inactivated virus particles provided significant protection from infection and high viremia. We examine a vaccine platform combining DNA and recombinant Env protein co-immunization at the same time to generate both strong cellular and humoral immune responses. Methods