Search

Developing a safe and effective preventive for HIV-1 remains the hope for controlling the global AIDS epidemic. Recently, mRNA vaccines have emerged as a promising alternative to conventional vaccine approaches, primarily due to their rapid development and potential for low-cost manufacture. Despite the advantages of mRNA vaccines, challenges remain, especially due to the adverse effects of the delivery vehicle and low delivery efficiency. As a result, Luna Labs is developing a short carbon nanotube-based delivery platform (NanoVac) that can co-deliver mRNA and HIV-1 glycoproteins to the immune system efficiently with negligible toxicity. Surface chemistries of NanoVac were optimized to guide antigen/mRNA loading density and presentation. Multiple formulations were engineered for compatibility with both intramuscular and intranasal administration. NanoVac candidates demonstrated immunogenicity in rabbits and generated human-derived humoral and cellular responses in humanized mice (HIS). Briefly, 33% of the HIV-1–infected HIS mice vaccinated with NanoVac–mRNA was cleared of virus infection by 8–weeks post-infection. Finally, NanoVac stabilized the loaded mRNA against degradation under refrigeration for at least three months, reducing the cold chain burden for vaccine deployment.

The HIV-1 envelope glycoprotein spike is the target of antibodies, and therefore represents the main viral antigen for antibody-based vaccine design. One of the challenges in HIV-1 vaccine development is finding efficient ways for the immune system to recognize and respond to HIV-1 without establishing an infection. Since HIV-1 enters the body at mucosal surfaces, induction of immune response at these sites is a preferred preventive approach. Nasal administration is a very effective route for mucosal immunization since it can stimulate mucosal immune responses both locally and distantly. In this paper, Luna develops a safe, short carbon nanotube (CNT)-based, needle-free delivery platform known as “CNTVac”. The size of short CNT was controlled to possess HIV-1 particle-like morphology (100-200 nm) capable of efficiently delivering a broad range of antigens intranasally. PEG-Lipid served as the antigen conformation protector and mucosal barrier penetration enhancer (Schematic Figure) was localized between V1V2 antigens, which caused highly enhanced local IgA and systemic antibody IgG responses in mice and rabbits. The short CNT incorporated with PEG-Lipid could not only serve as efficient delivery system but also reduce the amount of lipid usage in order to balance the vaccine dosage in order to eliminate the potential adverse effect. These data suggest a promising platform technology for vaccine delivery.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106–107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

The HIV vaccine field now recognizes the potential importance of generating polyfunctional antibodies (Abs). The only clinical HIV vaccine trial to date to show significant efficacy (RV144) found that reduced infection rates correlated with the level of nonneutralizing Abs specific for the V2 region of the envelope glycoprotein. We have conducted a comprehensive preclinical reverse vaccinology-based vaccine program that has included the design and production and testing of numerous scaffolded V2 region immunogens. The most immunogenic vaccine regimen in nonhuman primates among those studied as part of this program consisted of a cocktail of three immunogens presenting V2 from different viruses and clades in the context of different scaffolds. Presently we demonstrate that the V2-specific Ab response from this regimen was highly durable and functionally diverse for the duration of the study (25 weeks after the final immunization). The total IgG binding response at this late time point exhibited only an ∼5× reduction in potency. Three immunizations appeared essential for the elicitation of a strong Ab-dependent cellular cytotoxicity (ADCC) response for all animals, as opposed to the Ab-dependent cellular phagocytosis (ADCP) and virus capture responses, which were comparably potent after only 2 immunizations. All functionalities measured were highly durable through the study period. Therefore, testing this vaccine candidate for its protective capacity is warranted. IMPORTANCE The only HIV vaccine trial for which protective efficacy was detected correlated this efficacy with V2-specific Abs that were effectively nonneutralizing. This result has fueled a decade of HIV vaccine research focused on designing an HIV vaccine capable of eliciting V2-focused, polyfunctional Abs that effectively bind HIV and trigger various leukocytes to kill the virus and restrict viral spread. From the numerous vaccine candidates designed and tested as part of our V2-focused preclinical vaccine program, we have identified immunogens and a vaccine regimen that induces a highly durable and polyfunctional V2-focused Ab response in rhesus macaques, described herein.

Prophylactic HIV vaccines must elicit antibodies (Abs) against the virus envelope glycoproteins (Env) to effectively prevent HIV infection. We investigated a vaccine platform that utilizes immune complexes made of Env proteins gp120 and monoclonal Abs (mAbs) against different gp120 epitopes. We previously observed alterations in V3 antigenicity upon formation of certain gp120/mAb complexes and demonstrated the ability of these complexes to modulate the elicitation of V3 Ab responses. However, the effects on the V1V2 domain, an important target for Abs that correlate with vaccine-induced protection against HIV, have not been studied, nor have immune complex vaccines made with non-B subtype Env. This study compared subtypes B (JRFL) and CRF_01.AE (A244) Env gp120 proteins in complex with selected gp120-specific mAbs. Allosteric and antigenic changes were detected on these immune complexes, indicating that gp120/mAb interaction induces alterations on the Env surface that may modify the Env immunogenic properties. To evaluate this idea, mice were immunized with gp120/mAb complexes or their uncomplexed gp120 counterparts. The overall serum IgG titers elicited against gp120 were comparable, but a marked skewing toward V1V2 or V3 was evident and dependent on the gp120 strain and the specificity of the mAb used to form the complexes. Compared with uncomplexed gp120JRFL, gp120JRFL complexed with CD4bs or V1V2 mAbs, but not with C2 or V3 mAbs, elicited V3 Abs of greater titers and breadth, and Abs more capable of neutralizing tier 1 virus. Epitope mapping revealed a shift to a more conserved site in the V3 crown. However, the complexes did not enhance V1V2 Ab response, and the elicited V1V2 Abs were not cross-reactive. This profile contrasts with Ab responses to gp120A244/mAb complexes. Notably, gp120A244/mAb complexes induced higher levels of V1V2 Abs with some cross-reactivity, while also stimulating weak or strain-specific V3 Abs. Sera from gp120A244/mAb complex-immunized animals displayed no measurable virus neutralization but did mediate Ab-dependent cellular phagocytosis, albeit at levels similar to that induced by gp120A244 alone. These data indicate the potential utility of immune complexes as vaccines to shape Ab responses toward or away from Env sites of interest.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4β7, a gut-homing receptor. Using both cell-surface expressed α4β7 and a soluble α4β7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4β7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4β7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4β7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4β7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4β7. It includes the canonical LDV/I α4β7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4β7 interactions. These mAbs recognize conformations absent from the β- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4β7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

The V1V2 region of the HIV-1 Envelope is the target of several broadly neutralizing antibodies (bNAbs). RV144 vaccinees elicited binding antibodies to V1V2, which correlated with a reduced risk of HIV infection, yet these antibodies were without broad neutralizing activity. Antibodies targeting V2 also correlated with delayed infection and reduced viral load in immunized macaques challenged with SIV or SHIV. To focus immune responses on V1V2, we designed a panel of immunogens by engrafting the native, glycosylated V1V2 domain onto multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibody responses that targeted structurally distinct V1V2 epitopes. The plasma had neutralizing activity and was functionally active for ADCC and phagocytosis that was detectable 1-2 years after immunizations ended. This study demonstrates that multivalent V1V2-scaffold protein immunogens are differentially effective at inducing V1V2-targeted antibody responses with characteristics associated with protection.

The V1V2 region of the HIV-1 envelope is the target of several broadly neutralizing antibodies (bNAbs). Antibodies to V1V2 elicited in the RV144 clinical trial correlated with a reduced risk of HIV infection, but these antibodies were without broad neutralizing activity. Antibodies targeting V1V2 also correlated with a reduced viral load in immunized macaques challenged with simian immunodeficiency virus (SIV) or simian/human immunodeficiency virus (SHIV). To focus immune responses on V1V2, we engrafted the native, glycosylated V1V2 domain onto five different multimeric scaffold proteins and conducted comparative immunogenicity studies in macaques. Vaccinated macaques developed high titers of plasma and mucosal antibodies that targeted structurally distinct V1V2 epitopes. Plasma antibodies displayed limited neutralizing activity but were functionally active for ADCC and phagocytosis, which was detectable 1–2 years after immunizations ended. This study demonstrates that multivalent, glycosylated V1V2-scaffold protein immunogens focus the antibody response on V1V2 and are differentially effective at inducing poly-functional antibodies with characteristics associated with protection.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.

The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the β-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope. IMPORTANCE The correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

Strong antibody (Ab) responses against V1V2 epitopes of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) correlated with reduced infection rates in studies of HIV, simian-human immunodeficiency virus (SHIV), and simian immunodeficiency virus (SIV). In order to focus the Ab response on V1V2, we used six V1V2 sequences and nine scaffold proteins to construct immunogens which were tested using various immunization regimens for their ability to induce cross-reactive and biologically active V2 Abs in rabbits. A prime/boost immunization strategy was employed using gp120 DNA and various V1V2-scaffold proteins. The rabbit polyclonal Ab responses (i) were successfully focused on the V1V2 region, with weak or only transient responses to other Env epitopes, (ii) displayed broad cross-reactive binding activity with gp120s and the V1V2 regions of diverse strains from clades B, C, and E, (iii) included V2 Abs with specificities similar to those found in HIV-infected individuals, and (iv) remained detectable ≥1 year after the last boosting dose. Importantly, sera from rabbits receiving V1V2-scaffold immunogens displayed Ab-dependent cellular phagocytosis whereas sera from rabbits receiving only gp120 did not. The results represent the first fully successful example of reverse vaccinology in the HIV vaccine field with rationally designed epitope scaffold immunogens inducing Abs that recapitulate the epitope specificity and biologic activity of the human monoclonal Abs from which the immunogens were designed. Moreover, this is the first immunogenicity study using epitope-targeting, rationally designed vaccine constructs that induced an Fc-mediated activity associated with protection from infection with HIV, SIV, and SHIV. IMPORTANCE Novel immunogens were designed to focus the antibody response of rabbits on the V1V2 epitopes of HIV-1 gp120 since such antibodies were associated with reduced infection rates of HIV, SIV, and SHIV. The vaccine-induced antibodies were broadly cross-reactive with the V1V2 regions of HIV subtypes B, C and E and, importantly, facilitated Fc-mediated phagocytosis, an activity not induced upon immunization of rabbits with gp120. This is the first immunogenicity study of vaccine constructs that focuses the antibody response on V1V2 and induces V2-specific antibodies with the ability to mediate phagocytosis, an activity that has been associated with protection from infection with HIV, SIV, and SHIV.

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.

HIV-1 envelope (Env) antigen is an important target for both neutralizing and non-neutralizing antibodies (Ab) against the virus. In the RV144 vaccine trial, correlate for reduced risk of HIV acquisition is the presence of binding but non-neutralizing Ab to V1V2 and V3 loops of HIV Env gp120. However, induction of high titer, durable, and cross-reactive Ab responses against these Env regions by vaccination is still an elusive goal. Our past studies have shown that V3 immunogenicity can be significantly enhanced by vaccination with gp120/mAb immune complexes (IC) vs gp120 alone. Current study further examined parameters that confer enhanced immunogenicity to IC by testing in mice IC vaccines made with different HIV Env gp120 (clade B vs clade E) and mAb of different specificities (V3, V2, or CD4 binding site (CD4bs)). In addition, IC made of clade C gp140 and CD4bs mAb were also tested. The data showed that Greater titers of cross-reactive V3-binding Ab were induced by IC made of clade B gp120 and mAb to V2 or CD4bs as compare with gp120 alone, although the gp120/CD4bs IC was more potent in eliciting neutralizing V3 Ab. IC made with clade E gp120 or clade C gp140 did not show the same effects.Greater Ab responses to V1V2 were induced by IC made of clade E gp120 vs gp120 alone, regardless of the mAb used for IC, but the Ab were not neutralizing. IC made with clade B gp120 and clade C gp140 did not enhance V1V2 Ab titers. These results demonstrate the capacity of Env/mAb IC vaccines to modulate induction of Ab responses to V1V2 and V3, and this activity is dependent on both Env proteins and mAb used to form IC. Further exploration using a cocktail of IC is proposed to improve Ab repertoires generated upon vaccination.

RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in Escherichia coli . Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5′ end. Here we show that the preference of E. coli RNase E for 5′-monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis , indicating that the 5′-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5′-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli . Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced sixfold relative to wild-type RNase E levels.

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

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.

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

Binding of the third variable region (V3) of the HIV-1 envelope glycoprotein gp120 to the cell-surface coreceptors CCR5 or CXCR4 during viral entry suggests that there are conserved structural elements in this sequence-variable region. These conserved elements could serve as epitopes to be targeted by a vaccine against HIV-1. Here we perform a systematic structural analysis of representative human anti-V3 monoclonal antibodies in complex with V3 peptides, revealing that the crown of V3 has four conserved structural elements: an arch, a band, a hydrophobic core and the peptide backbone. These are either unaffected by or are subject to minimal sequence variation. As these regions are targeted by cross-clade neutralizing human antibodies, they provide a blueprint for the design of vaccine immunogens that could elicit broadly cross-reactive protective antibodies.

Background Experiments were designed based on the hypothesis that recombinant vaccine constructs can focus the immune response on shared HIV-1 neutralizing epitopes, and that if not diverted by other biologically irrelevant epitopes, high titers of cross-clade neutralizing antibodies (ccNAbs) will be induced. Indeed, previous experiments showed that when the immune response was focused on V3, ccNAbs were induced (Zolla-Pazner et al, Virology, 2008).

Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modelled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67 and Lys-112 [corrected], is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The side-chains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67 and Lys-112 [corrected], which probably enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. In contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth or ribonuclease activity.

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of theEscherichia coliferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypicEscherichia colisiderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.

Background The V3 loop is a major neutralizing determinant of the HIV-1 virus. Cholera toxin subunit B (CTB) is a highly immunogenic protein and it has been used in fusion constructs to enhance immunogenicity of target proteins. We hypothesized that a rationally designed immunogen, based on V3 and CTB, could induce high titers of Abs with binding mode and epitope specificity similar to known broadly neutralizing anti-V3 mAbs.

Corrigendum to “Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold” [Virology 351 (2010) 513–523] Maxim Totrov ⁎, Xunqing Jiang , Xiang-Peng Kong , Sandra Cohen , Chavdar Krachmarov , Aidy Salomon , Constance Williams , Michael S. Seaman , Ruben Abagyan , Timothy Cardozo , Miroslaw K. Gorny , Shixia Wang , Shan Lu , Abraham Pinter , Susan Zolla-Pazner b,c,⁎,2

Background Skin is an excellent site for vaccine administration due to the abundance of antigen presenting cells and easy access. When DNA or protein is delivered into the skin, uptake and expression of immunogens can lead to a rapid and robust induction of immune responses.Skin tattooing has been suggested as a useful tool delivering vaccines intradermally. We have chosen to use the skin tattooing technique to deliver both DNA and protein immunogens that are focusing the immune response on the highly immunogenic V3 region of HIV-1 gp120. Methods A GFP-containing plasmid was used first to optimize the protocol for skin tattooing on BALB/c mice. Subsequently, BALB/c mice were immunized using a DNA prime/Protein boost regimen. DNA skin tattooing was used first as the prime to deliver a gp120-encoding DNA plasmid intradermally at weeks 0, 2, and 4. The animals were then given a fusion protein, V3-bearing cholera toxin subunit B (V3-CTB), via skin tattooing (Group1; n=5) or intramuscular injection (Group2; n=5) at weeks 10 and 14. Two weeks after the final immunization, the sera were collected and analyzed. Results GFP expressed in skin cells was visualized by confocal microscopy showing that skin tattooing can effectively deliver DNA into these cells. ELISA assays using sera drawn after the final immunization confirmed that tattoodelivered DNA followed by V3-CTBelicited V3 binding antibodies, and these antibodies have potent neutralizing activities in an HIV pseudovirus neutralization assay. Conclusion Immunization by skin tattooing is an easy, effective, and economical way to administer both DNA and protein vaccines. Interestingly, the needle perforations may serve as a potent and natural adjuvant for the immunization, making skin tattooing a promising vaccine delivery technique.