Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp... more Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp41 int folds into a compact elongated structure that induces neutralizing antibodies upon immunization. Conclusion: Presentation of gp41 int in a lipid environment is beneficial to induce neutralizing antibodies. Significance: Membrane-anchored gp41 int is a promising antigen to improve breadth and potency of anti-gp41 antibody responses. * This work was supported in part by the Bill and Melinda Gates Foundationfunded Consortium of AIDS Vaccine Discovery (Grant 38637), the Comprehensive Antibody-Vaccine Immune Monitoring Consortium (Grant 38619), and Sidaction (to W. W.).
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2012
Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Althou... more Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful 1-3 , a minority of individuals naturally develop these antibodies after many years of infection 4-7. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the Correspondence should be addressed to L.M.
Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glyc... more Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.
BaCLeroidesfragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intesti... more BaCLeroidesfragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intestinal microbiota. It is, however, an opportunistic pathogen and has been ranked as the most prevalent isolate in cases of anaerobic septicaemia. Similar to most bacteria, ammonium is assimilated in B. fragilis through the action of glutamine synthetase (GS). Glutamine is vital to nitrogen metabolism as it serves as a precursor to many secondary metabolites. GS enzymes are, therefore, vital to the growth of the organism and many prokaryotes are known to possess two or more isofonns of the enzyme. In addition, GS expression and regulation is usually tightly regulated in concert with the availability of nitrogen. Previous studies have identified a single GSIII encoding gene (ginN) in B. fragilis. In this dissertation, an additional ORF coding for a putative GSI enzyme in B. fragilis was identified, isolated and functionally characterized. A putative regulatory protein was also identified and its functional contribution to nitrogen metabolism was determined, in order to extend our understanding of nitrogen assimilation in B.fragilis. In order to evaluate ifglnN was the only functional GS encoding gene in B.fragilis, a mutant strain containing an insertional inactivation of the ginN gene, B. jragilis 638QglnN, was constructed. Physiological analyses demonstrated that B. jragilis 638QglnN was able to grow in minimal medium supplemented with elevated concentrations of ammonium chloride (NH 4 CI) as the only nitrogen source. Residual GS activity was also detected in B. fragilis 638QglnN grown in medium with both ammonium shortage and excess. These findings suggested that an additional gene in B. jragilis, may encode a functional GS, explaining the GS activity and growth of the bacterium in high ammonium-containing medium. Bioinformatic analyses lead to the identification of an ORF encoding a putative ginA gene in the B. fragilis genome. The gene was found to have a low amino acid identity to other characterized GSI enzymes from different bacteria, however, it contained three out of the five highly conserved GS regions. This gene was cloned into an E. coli glutamine auxotroph. Although RNA hybridization experiments showed that the gene was transcribed in E. coli, it failed to complement the glutamine auxotrophy of this strain. Another ORF coding for a putative GS regulatory protein was also identified in B.fragilis. This gene was located directly downstream of ginN and was annotated as belonging to the
Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Anti... more Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Antibody 10E8, reactive with the distal portion of the membrane-proximal external region (MPER) of HIV-1 gp41, is broadly neutralizing. However, the ontogeny of distal MPER antibodies and the relationship of memory B cell to plasma bnAbs are poorly understood. HIV-1-specific memory B cell flow sorting and proteomic identification of anti-MPER plasma antibodies from an HIV-1-infected individual were used to isolate broadly neutralizing distal MPER bnAbs of the same B cell clonal lineage. Structural analysis demonstrated that antibodies from memory B cells and plasma recognized the envelope gp41 bnAb epitope in a distinct orientation compared with other distal MPER bnAbs. The unmutated common ancestor of this distal MPER bnAb was autoreactive, suggesting lineage immune tolerance control. Construction of chimeric antibodies of memory B cell and plasma antibodies yielded a bnAb that potently neu...
Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier... more Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.
The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a gl... more The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following in...
<p>The sensitivity of nine CAP257 clones from 7, 30, 54, 93, and 174 weeks p.i. to broadly ... more <p>The sensitivity of nine CAP257 clones from 7, 30, 54, 93, and 174 weeks p.i. to broadly neutralizing antibodies targeting the four known sites of vulnerability was measured in a TZM-bl assay. The timing of wave 1 (red), wave 2 (green), and wave 3 (brown) neutralization is summarized above with arrows. Increases in IC<sub>50</sub> relative to the 7 week clone are colored as follows: 5–10 fold (yellow), >10 fold (orange), complete neutralization resistance (red).</p
The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequentl... more The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs b...
American journal of physiology. Regulatory, integrative and comparative physiology, 2003
Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mech... more Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mechanisms by which leptin levels are increased may be species specific, however, with some species upregulating adipose leptin production and others expressing leptin in the placenta. Placental leptin expression was examined in representative species of the two most abundant mammalian orders, Rodentia and Chiroptera, and in cultured human choriocarcinoma (BeWo) cells. Leptin mRNA was expressed in BeWo cells and in placentas of Myotis lucifugus (little brown bat), Eptesicus fuscus (big brown bat), and Rattus norvegicus (laboratory rat), but not the common laboratory mouse Mus musculus. cAMP stimulated secretion of leptin from BeWo cells and also stimulated leptin mRNA expression in the cells. In addition to adipose and placental tissue, leptin transcript in M. lucifugus was detectable in heart, spleen, and liver, but not in lung, brain, and kidney. Hepatic expression was also observed in E. ...
Introduction HIV-1 subtype C accounts for most of the global AIDS epidemic, yet most drugs agains... more Introduction HIV-1 subtype C accounts for most of the global AIDS epidemic, yet most drugs against HIV-1 are based on subtype B. In this study, we mapped the binding site (bs) and determined the molecular interactions between UCLA1 aptamer and consensus HIV-1 subtype C (ConC) gp120. Methods RNA aptamer, named UCLA1, which broadly neutralized HIV-1 including subtype C Env-pseudotyped viruses, was bound to ConC gp120 using the BIAcore. The aptamer was also bound to truncated (∆V1/V2, ∆V3) and mutated (I420R – co-receptor (CoR) bs, D368R - CD4 bs) ConC gp120. The amino acid residues that interact with the aptamer were determined using a panel of single site-directed point mutations on gp120 of ConC Env-pseudotyped viruses. Mutated viruses were respectively tested for resistance to the aptamer. Results The binding kinetics of the aptamer to ConC gp120 showed a KD of 0.28 ±0.04nM. The binding profile of the aptamer to ConC ∆V1/V2 gp120 was observed to be lower than binding to ∆V3 and wil...
The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and ... more The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.
Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these a... more Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.
l e t t e r s nature medicine advance online publication Neutralizing antibodies are likely to pl... more l e t t e r s nature medicine advance online publication Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful 1-3 , a minority of individuals naturally develop these antibodies after many years of infection 4-7 . How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strainspecific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp... more Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp41 int folds into a compact elongated structure that induces neutralizing antibodies upon immunization. Conclusion: Presentation of gp41 int in a lipid environment is beneficial to induce neutralizing antibodies. Significance: Membrane-anchored gp41 int is a promising antigen to improve breadth and potency of anti-gp41 antibody responses.
Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp... more Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp41 int folds into a compact elongated structure that induces neutralizing antibodies upon immunization. Conclusion: Presentation of gp41 int in a lipid environment is beneficial to induce neutralizing antibodies. Significance: Membrane-anchored gp41 int is a promising antigen to improve breadth and potency of anti-gp41 antibody responses. * This work was supported in part by the Bill and Melinda Gates Foundationfunded Consortium of AIDS Vaccine Discovery (Grant 38637), the Comprehensive Antibody-Vaccine Immune Monitoring Consortium (Grant 38619), and Sidaction (to W. W.).
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2012
Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Althou... more Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful 1-3 , a minority of individuals naturally develop these antibodies after many years of infection 4-7. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the Correspondence should be addressed to L.M.
Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glyc... more Background: An ongoing challenge in HIV-1 vaccine research is finding a novel HIV-1 envelope glycoprotein (Env)-based immunogen that elicits broadly cross-neutralizing antibodies (bnAbs) without requiring complex sequential immunization regimens to drive the required antibody affinity maturation. Previous vaccination studies have shown monomeric Env and Env trimers which contain the GCN4 leucine zipper trimerization domain and are covalently bound to the first two domains of CD4 (2dCD4S60C) generate potent bnAbs in small animals. Since SOSIP.664 trimers are considered the most accurate, conformationally intact representation of HIV-1 Env generated to date, this study further evaluated the immunogenicity of SOSIP.664 HIV Env trimers (the well characterized BG505 and FVCEnv) covalently complexed to 2dCD4S60C. Methods: Recombinant BG505 SOSIP.664 and FVCEnv SOSIP.664 were expressed in mammalian cells, purified, covalently coupled to 2dCD4S60C and antigenically characterized for their interaction with HIV-1 bnAbs. The immunogenicity of BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C was investigated in New Zealand white rabbits and compared to unliganded FVCEnv and 2dCD4S60C. Rabbit sera were tested for the presence of neutralizing antibodies against a panel of 17 pseudoviruses. Results: Both BG505 SOSIP.664-2dCD4S60C and FVCEnv SOSIP.664-2dCD4S60C elicited a potent, HIV-specific response in rabbits with antibodies having considerable potency and breadth (70.5% and 76%, respectively) when tested against a global panel of 17 pseudoviruses mainly composed of harder-to-neutralize multiple clade tier-2 pseudoviruses. Conclusion: BG505 SOSIP.664-2dCD4S60C and FVCEnvSOSIP.664-2dCD4S60C are highly immunogenic and elicit potent, broadly neutralizing antibodies, the extent of which has never been reported previously for SOSIP.664 trimers. Adding to our previous results, the ability to consistently elicit these types of potent, cross-neutralizing antibody responses is dependent on novel epitopes exposed following the covalent binding of Env (independent of sequence and conformation) to 2dCD4S60C. These findings justify further investment into research exploring modified open, CD4-bound Env conformations as novel vaccine immunogens.
BaCLeroidesfragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intesti... more BaCLeroidesfragilis is a gram-negative, non spore-forming, obligate anaerobe of the human intestinal microbiota. It is, however, an opportunistic pathogen and has been ranked as the most prevalent isolate in cases of anaerobic septicaemia. Similar to most bacteria, ammonium is assimilated in B. fragilis through the action of glutamine synthetase (GS). Glutamine is vital to nitrogen metabolism as it serves as a precursor to many secondary metabolites. GS enzymes are, therefore, vital to the growth of the organism and many prokaryotes are known to possess two or more isofonns of the enzyme. In addition, GS expression and regulation is usually tightly regulated in concert with the availability of nitrogen. Previous studies have identified a single GSIII encoding gene (ginN) in B. fragilis. In this dissertation, an additional ORF coding for a putative GSI enzyme in B. fragilis was identified, isolated and functionally characterized. A putative regulatory protein was also identified and its functional contribution to nitrogen metabolism was determined, in order to extend our understanding of nitrogen assimilation in B.fragilis. In order to evaluate ifglnN was the only functional GS encoding gene in B.fragilis, a mutant strain containing an insertional inactivation of the ginN gene, B. jragilis 638QglnN, was constructed. Physiological analyses demonstrated that B. jragilis 638QglnN was able to grow in minimal medium supplemented with elevated concentrations of ammonium chloride (NH 4 CI) as the only nitrogen source. Residual GS activity was also detected in B. fragilis 638QglnN grown in medium with both ammonium shortage and excess. These findings suggested that an additional gene in B. jragilis, may encode a functional GS, explaining the GS activity and growth of the bacterium in high ammonium-containing medium. Bioinformatic analyses lead to the identification of an ORF encoding a putative ginA gene in the B. fragilis genome. The gene was found to have a low amino acid identity to other characterized GSI enzymes from different bacteria, however, it contained three out of the five highly conserved GS regions. This gene was cloned into an E. coli glutamine auxotroph. Although RNA hybridization experiments showed that the gene was transcribed in E. coli, it failed to complement the glutamine auxotrophy of this strain. Another ORF coding for a putative GS regulatory protein was also identified in B.fragilis. This gene was located directly downstream of ginN and was annotated as belonging to the
Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Anti... more Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Antibody 10E8, reactive with the distal portion of the membrane-proximal external region (MPER) of HIV-1 gp41, is broadly neutralizing. However, the ontogeny of distal MPER antibodies and the relationship of memory B cell to plasma bnAbs are poorly understood. HIV-1-specific memory B cell flow sorting and proteomic identification of anti-MPER plasma antibodies from an HIV-1-infected individual were used to isolate broadly neutralizing distal MPER bnAbs of the same B cell clonal lineage. Structural analysis demonstrated that antibodies from memory B cells and plasma recognized the envelope gp41 bnAb epitope in a distinct orientation compared with other distal MPER bnAbs. The unmutated common ancestor of this distal MPER bnAb was autoreactive, suggesting lineage immune tolerance control. Construction of chimeric antibodies of memory B cell and plasma antibodies yielded a bnAb that potently neu...
Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier... more Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1) viruses or rare cases of vaccine-matched neutralization-resistant (tier-2) viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER) that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.
The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a gl... more The development of biomedical interventions to reduce acquisition of HIV-1 infection remains a global priority, however their potential effectiveness is challenged by very high HIV-1 envelope diversity. Two large prophylactic trials in high incidence, clade C epidemic regions in southern Africa are imminent; passive administration of the monoclonal antibody VRC01, and active immunization with a clade C modified RV144-like vaccines. We have created a large representative panel of C clade viruses to enable assessment of antibody responses to vaccines and natural infection in Southern Africa, and we investigated the genotypic and neutralization properties of recently transmitted clade C viruses to determine how viral diversity impacted antibody recognition. We further explore the implications of these findings for the potential effectiveness of these trials. A panel of 200 HIV-1 Envelope pseudoviruses was constructed from clade C viruses collected within the first 100 days following in...
<p>The sensitivity of nine CAP257 clones from 7, 30, 54, 93, and 174 weeks p.i. to broadly ... more <p>The sensitivity of nine CAP257 clones from 7, 30, 54, 93, and 174 weeks p.i. to broadly neutralizing antibodies targeting the four known sites of vulnerability was measured in a TZM-bl assay. The timing of wave 1 (red), wave 2 (green), and wave 3 (brown) neutralization is summarized above with arrows. Increases in IC<sub>50</sub> relative to the 7 week clone are colored as follows: 5–10 fold (yellow), >10 fold (orange), complete neutralization resistance (red).</p
The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequentl... more The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs b...
American journal of physiology. Regulatory, integrative and comparative physiology, 2003
Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mech... more Increased plasma level of leptin appears to be a ubiquitous feature of pregnant mammals. The mechanisms by which leptin levels are increased may be species specific, however, with some species upregulating adipose leptin production and others expressing leptin in the placenta. Placental leptin expression was examined in representative species of the two most abundant mammalian orders, Rodentia and Chiroptera, and in cultured human choriocarcinoma (BeWo) cells. Leptin mRNA was expressed in BeWo cells and in placentas of Myotis lucifugus (little brown bat), Eptesicus fuscus (big brown bat), and Rattus norvegicus (laboratory rat), but not the common laboratory mouse Mus musculus. cAMP stimulated secretion of leptin from BeWo cells and also stimulated leptin mRNA expression in the cells. In addition to adipose and placental tissue, leptin transcript in M. lucifugus was detectable in heart, spleen, and liver, but not in lung, brain, and kidney. Hepatic expression was also observed in E. ...
Introduction HIV-1 subtype C accounts for most of the global AIDS epidemic, yet most drugs agains... more Introduction HIV-1 subtype C accounts for most of the global AIDS epidemic, yet most drugs against HIV-1 are based on subtype B. In this study, we mapped the binding site (bs) and determined the molecular interactions between UCLA1 aptamer and consensus HIV-1 subtype C (ConC) gp120. Methods RNA aptamer, named UCLA1, which broadly neutralized HIV-1 including subtype C Env-pseudotyped viruses, was bound to ConC gp120 using the BIAcore. The aptamer was also bound to truncated (∆V1/V2, ∆V3) and mutated (I420R – co-receptor (CoR) bs, D368R - CD4 bs) ConC gp120. The amino acid residues that interact with the aptamer were determined using a panel of single site-directed point mutations on gp120 of ConC Env-pseudotyped viruses. Mutated viruses were respectively tested for resistance to the aptamer. Results The binding kinetics of the aptamer to ConC gp120 showed a KD of 0.28 ±0.04nM. The binding profile of the aptamer to ConC ∆V1/V2 gp120 was observed to be lower than binding to ∆V3 and wil...
The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and ... more The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.
Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these a... more Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.
l e t t e r s nature medicine advance online publication Neutralizing antibodies are likely to pl... more l e t t e r s nature medicine advance online publication Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful 1-3 , a minority of individuals naturally develop these antibodies after many years of infection 4-7 . How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strainspecific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp... more Background: HIV-1 gp41 MPER is a target for inducing broadly neutralizing antibodies. Results: Gp41 int folds into a compact elongated structure that induces neutralizing antibodies upon immunization. Conclusion: Presentation of gp41 int in a lipid environment is beneficial to induce neutralizing antibodies. Significance: Membrane-anchored gp41 int is a promising antigen to improve breadth and potency of anti-gp41 antibody responses.
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Papers by Nancy Tumba