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1.  Regional Clustering of Shared Neutralization Determinants on Primary Isolates of Clade C Human Immunodeficiency Virus Type 1 from South Africa 
Journal of Virology  2002;76(5):2233-2244.
Clade C is one of the most prevalent genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in the world today and one of the least studied with respect to neutralizing antibodies. Most information on HIV-1 serology as it relates to neutralization is derived from clade B. Clade C primary isolates of HIV-1 from South Africa and Malawi were shown here to resemble clade B isolates in their resistance to inhibition by soluble CD4 and their sensitivity to neutralization by human monoclonal antibody immunoglobulin G1b12 and, to a lesser extent, 2F5. Unlike clade B isolates, however, all 16 clade C isolates examined resisted neutralization by 2G12. Infection with clade C HIV-1 in a cohort of female sex workers in South Africa generated antibodies that neutralized the autologous clade C isolate and T-cell-line-adapted (TCLA) strains of clade B. Neutralization of clade B TCLA strains was much more sensitive to the presence of autologous gp120 V3 loop peptides compared to the neutralization of clade C isolates in most cases. Thus, the native structure of gp120 on primary isolates of clade C will likely pose a challenge for neutralizing antibody induction by candidate HIV-1 vaccines much the same as it has for clade B. The autologous neutralizing antibody response following primary infection with clade C HIV-1 in South Africa matured slowly, requiring at least 4 to 5 months to become detectable. Once detectable, extensive cross-neutralization of heterologous clade C isolates from South Africa was observed, suggesting an unusual degree of shared neutralization determinants at a regional level. This high frequency of cross-neutralization differed significantly from the ability of South African clade C serum samples to neutralize clade B isolates but did not differ significantly from results of other combinations of clade B and C reagents tested in checkerboard assays. Notably, two clade C serum samples obtained after less than 2 years of infection neutralized a broad spectrum of clade B and C isolates. Other individual serum samples showed a significant clade preference in their neutralizing activity. Our results suggest that clades B and C are each comprised of multiple neutralization serotypes, some of which are more clade specific than others. The clustering of shared neutralization determinants on clade C primary HIV-1 isolates from South Africa suggests that neutralizing antibodies induced by vaccines will have less epitope diversity to overcome at a regional level.
PMCID: PMC135941  PMID: 11836401
2.  Identification and Characterization of a New Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody 
Journal of Virology  2004;78(17):9233-9242.
The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.
PMCID: PMC506938  PMID: 15308718
3.  Prophylactic and Therapeutic Efficacy of Human Monoclonal Antibodies against H5N1 Influenza 
PLoS Medicine  2007;4(5):e178.
New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.
Methods and Findings
Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).
These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.
Cameron Simmons and colleagues provide proof of concept that human monoclonal antibodies with neutralizing activity can be rapidly generated from peripheral blood of convalescent patients and are effective in preventing and treating H5N1 infection in a mouse model.
Editors' Summary
Every year, millions of people catch influenza, a viral disease of the nose, throat, and airways. Although most recover, influenza outbreaks (epidemics) kill about half a million people annually. Epidemics occur because small but frequent changes in the viral proteins (antigens) to which the immune system responds mean that an immune response produced one year provides only partial protection against influenza the next year. Human flu viruses also occasionally appear that contain major antigenic changes. People have little or no immunity to such viruses (which often originate in animals or birds), so these viruses can start deadly pandemics—global epidemics. The Spanish flu pandemic in 1918/9, Asian flu in 1957, and Hong Kong flu in 1968 all killed millions. Experts believe that another pandemic is overdue and may be triggered by the avian H5N1 influenza virus—the name indicates that this bird virus carries type 5 hemagglutinin and type 1 neuraminidase, the two major flu antigens. H5N1, which rapidly kills infected birds, is now present in flocks around the world and, since 1997, it has caused 258 cases of human flu and 153 deaths. People have caught H5N1 through close contact with infected birds but, luckily, H5N1 rarely passes between people.
Why Was This Study Done?
H5N1 might acquire the ability to move between people and start a human influenza pandemic at any time. Some of the H5N1 viruses are resistant to the antiviral drugs used to treat flu and there will inevitably be a lag of some months between the emergence of a human pandemic H5N1 strain and the bulk production of a vaccine effective against it. Thus, new preventative and therapeutic strategies are needed to combat human infections with H5N1. One possibility is passive immunotherapy—treating people with antibodies (proteins that recognize antigens) that can stop H5N1 from infecting cells (so-called neutralizing antibodies). In this study, the researchers have generated neutralizing human monoclonal antibodies (laboratory-produced preparations that contain one type of human antibody) and tested their ability to halt viral growth in mice infected with H5N1.
What Did the Researchers Do and Find?
Patients who have survived infection with H5N1 make neutralizing antibodies, so the researchers isolated and immortalized the immune cells making these antibodies from the patients' blood. They grew up each cell separately and purified the antibody that the cells made. These monoclonal antibodies were then tested for their ability to neutralize H5N1 and other flu viruses in the laboratory. The researchers identified several that neutralized the H5N1 strain with which the patients were originally infected and chose two for further study. In the test tube, the four antibodies neutralized closely related H5N1 viruses and an H5N1 virus from a different lineage (clade) that has also caused human disease, in addition to the original H5N1 virus, although with different efficacies. In mice, the antibodies provided protection from infection with the original virus when given a day before or one to three days after infection. Three antibodies also partly protected the mice against H5N1 from a different clade. Finally, the researchers showed that the antibodies protected mice by limiting viral replication, by lessening the deleterious effects of the virus in the lungs, and by stopping viral spread out of the lungs.
What Do These Findings Mean?
These results indicate that passive immunotherapy with human monoclonal antibodies could help to combat avian H5N1 if (or when) it starts a human pandemic. Passive immunotherapy is already used to prevent infections with several other viruses. In addition, a crude form of the approach—early treatment of patients with plasma (the liquid portion of blood) from convalescent patients—halved the death rate during the Spanish flu pandemic. Large amounts of pure monoclonal antibodies can be relatively easily made for clinical use, and this study indicates that some monoclonal antibodies neutralize H5N1 viruses from different clades. The researchers sound a note of caution, however: Before passive immunotherapy can help to halt an H5N1 pandemic, they warn, the monoclonal antibodies will have to be tested to see whether they can neutralize not only all the currently circulating H5N1 viruses but also any emerging pandemic versions, which might be antigenically distinct.
Additional Information.
Please access these Web sites via the online version of this summary at
US Centers for Disease Control and Prevention information about influenza for patients and professionals including key facts about avian influenza
US National Institute of Allergy and Infectious Disease feature on seasonal, avian, and pandemic flu
World Health Organization factsheet on influenza and information on avian influenza, including latest figures for confirmed human cases
UK Health Protection Agency information on seasonal, avian, and pandemic influenza
Wikipedia pages on passive immunity and monoclonal antibodies (note: Wikipedia is an online encyclopedia that anyone can edit)
PMCID: PMC1880850  PMID: 17535101
4.  Correlation between circulating HIV-1 RNA and broad HIV-1 neutralizing antibody activity 
To examine the relationship between HIV-1 antigenic load (plasma RNA copies/ml) and broad HIV-1 neutralizing antibody activity.
Plasma from 120 HIV-1 infected patients, including HIV-1 Natural Viral Suppressors (similar to Elite Controllers), was tested for neutralization against 15 Tier 1/Tier 2 HIV-1 pseudoviruses. Broad HIV-1 neutralizing antibody activity was confirmed with IgG and heterlogous clade testing (18 pseudoviruses from Clades A, C, and CRF02_AG). Statistical analysis was performed to determine factors associated with broad HIV-1 neutralizing antibody activity.
Ten individuals with broad HIV-1 neutralizing antibody activity were identified. These individuals had a median CD4 count of 589 cells/ul (range 202–927), 1,611 HIV-1 RNA copies/ml (range 110–8,964), and 13 years since HIV diagnosis (range 1–22). There was a significant correlation between the presence of broadly neutralizing antibodies in those with HIV-1 RNA between 100 and 10,000 copies/ml compared to those <100 or >10,000 copies/ml (p=.0003 and .0245, respectively). Individuals with HIV-1 RNA 100–10,000 copies/ml had a higher number of Tier 2 viruses neutralized compared to the <100or >10,000 copies/ml groups (p=< .0001 and p=.076, respectively). Male sex was associated with broad HIV-1 neutralizing antibody activity (p=.016).
These results indicate that low but persistent HIV antigen expression correlates with broad HIV-1 neutralizing antibody activity. At higher levels of plasma viremia, neutralization titers were diminished. Conversely, at lower levels, there appears to be insufficient antigen stimulation to maintain high neutralization titers. These findings may have important implications in furthering the understanding of the humoral response to HIV infection.
PMCID: PMC3110998  PMID: 21283016
HIV; broadly neutralizing antibody; neutralizing activity; HIV RNA; natural viral suppressor; elite controller
5.  Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies 
Journal of Virology  2004;78(23):13232-13252.
Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+ plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+ plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade.
PMCID: PMC524984  PMID: 15542675
6.  Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies 
Journal of Virology  2005;79(16):10108-10125.
Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.
PMCID: PMC1182643  PMID: 16051804
7.  Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals 
PLoS ONE  2010;5(1):e8805.
The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.
Methods and Findings
We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.
This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.
PMCID: PMC2808385  PMID: 20098712
8.  Soluble HIV-1 Envelope Immunogens Derived from an Elite Neutralizer Elicit Cross-Reactive V1V2 Antibodies and Low Potency Neutralizing Antibodies 
PLoS ONE  2014;9(1):e86905.
We evaluated four gp140 Envelope protein vaccine immunogens that were derived from an elite neutralizer, subject VC10042, whose plasma was able to potently neutralize a wide array of genetically distinct HIV-1 isolates. We sought to determine whether soluble Envelope proteins derived from the viruses circulating in VC10042 could be used as immunogens to elicit similar neutralizing antibody responses by vaccination. Each gp140 was tested in its trimeric and monomeric forms, and we evaluated two gp140 trimer vaccine regimens in which adjuvant was supplied at all four immunizations or at only the first two immunizations. Interestingly, all four Envelope immunogens elicited high titers of cross-reactive antibodies that recognize the variable regions V1V2 and are potentially similar to antibodies linked with a reduced risk of HIV-1 acquisition in the RV144 vaccine trial. Two of the four immunogens elicited neutralizing antibody responses that neutralized a wide array of HIV-1 isolates from across genetic clades, but those responses were of very low potency. There were no significant differences in the responses elicited by trimers or monomers, nor was there a significant difference between the two adjuvant regimens. Our study identified two promising Envelope immunogens that elicited anti-V1V2 antibodies and broad, but low potency, neutralizing antibody responses.
PMCID: PMC3900663  PMID: 24466285
9.  The V1/V2 Domain of gp120 Is a Global Regulator of the Sensitivity of Primary Human Immunodeficiency Virus Type 1 Isolates to Neutralization by Antibodies Commonly Induced upon Infection 
Journal of Virology  2004;78(10):5205-5215.
A major problem hampering the development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) is the resistance of many primary viral isolates to antibody-mediated neutralization. To identify factors responsible for this resistance, determinants of the large differences in neutralization sensitivities of HIV-1 pseudotyped with Env proteins derived from two prototypic clade B primary isolates were mapped. SF162 Env pseudotypes were neutralized very potently by a panel of sera from HIV-infected individuals, while JR-FL Env pseudotypes were neutralized by only a small fraction of these sera. This differential sensitivity to neutralization was also observed for a number of monoclonal antibodies (MAbs) directed against sites in the V2, V3, and CD4 binding domains, despite often similar binding affinities of these MAbs towards the two soluble rgp120s. The neutralization phenotypes were switched for chimeric Envs in which the V1/V2 domains of these two sequences were exchanged, indicating that the V1/V2 region regulated the overall neutralization sensitivity of these Envs. These results suggested that the inherent neutralization resistance of JR-FL, and presumably of related primary isolates, is to a great extent mediated by gp120 V1/V2 domain structure rather than by sequence variations at the target sites. Three MAbs (immunoglobulin G-b12, 2G12, and 2F5) previously reported to possess broad neutralizing activity for primary HIV-1 isolates neutralized JR-FL virus at least as well as SF162 virus and were not significantly affected by the V1/V2 domain exchanges. The rare antibodies capable of neutralizing a broad range of primary isolates thus appeared to be targeted to exceptional epitopes that are not sensitive to V1/V2 domain regulation of neutralization sensitivity.
PMCID: PMC400352  PMID: 15113902
10.  Enhancing Exposure of HIV-1 Neutralization Epitopes through Mutations in gp41 
PLoS Medicine  2008;5(1):e9.
The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env) variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes.
Methods and Findings
Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41) of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced neutralization sensitivity exceeded 1,000-fold in several cases.
Two amino acid mutations within gp41 were identified that expose multiple discontinuous neutralization epitopes on diverse HIV-1 Env proteins. These exposed epitopes were shielded on the unmodified viral Env proteins, and several of the exposed epitopes encompass desired target regions for protective antibodies. Env proteins containing these modifications could act as a scaffold for presentation of such conserved domains, and may aid in developing methods to target antibodies to such regions.
Julie Overbaugh and colleagues analyze an HIV strain with high susceptibility to antibody neutralization and identify two gp41 envelope mutations that confer this sensitivity by exposing multiple neutralization epitopes.
Editors' Summary
In 1984 when scientists identified human immunodeficiency virus (HIV)—the cause of acquired immunodeficiency syndrome (AIDS)—many experts believed that a vaccine against HIV infection would soon be developed. Nearly 25 years later, there is still no such vaccine and with about 2.5 million new HIV infections in 2007, an effective vaccine is urgently needed to contain the AIDS epidemic. Vaccines provide protection against infectious diseases by priming the immune system to deal quickly and effectively with viruses and other pathogens. Vaccines do this by exposing the immune system to an immunogen—a fragment or harmless version of the pathogen. The immune system mounts a response against the immunogen and also “learns” from this experience so that if it is ever challenged with a virulent version of the same pathogen, it can quickly contain the threat. Many vaccines work by stimulating an antibody response. Antibodies are proteins made by the immune system that bind to molecules called antigens on the surface of pathogens. Antibodies that inactivate the invader upon binding to it are called “neutralizing” antibodies.
Why Was This Study Done?
Several characteristics of HIV have hampered the development of an effective vaccine. An “envelope” protein consisting of two subunits called gp120 and gp41 covers the outside of HIV. Many regions of this protein change rapidly, so the antibody response stimulated by a vaccine containing the envelope protein of one HIV variant provides little protection against other variants. However, other regions of the protein rarely change, so a vaccine that stimulates the production of antibodies to these “conserved” regions is likely to provide protection against many HIV variants. That is, it will stimulate the production of broadly neutralizing antibodies. Unfortunately, it has been difficult to find HIV vaccines that do this, because these conserved regions are often hidden from the immune system by other parts of the envelope protein. In this study, the researchers investigate the envelope protein of an HIV-1 variant they have isolated that is highly susceptible to inactivation by antibodies specific for these conserved regions. Comparing the envelope protein of this sensitive virus to closely related envelope proteins that are resistant to neutralization could identify features that might, if included in an envelope protein immunogen, produce a vaccine capable of generating broadly neutralizing antibodies.
What Did the Researchers Do and Find?
The researchers isolated a subtype A HIV-1 variant from a newly infected woman in Kenya that was efficiently neutralized by monoclonal antibodies (antibodies made by cells that have been cloned in the laboratory). These antibodies were specific for several different conserved regions of gp41 and gp120. The isolate was also neutralized by antibodies in blood from HIV-1-infected people. The envelope protein of the sensitive variant was the same as that of a resistant variant isolated at the same time from the woman, except for four amino acid changes in conserved regions of gp41 (proteins are made from long strings of amino acids). Using a technique called site-directed mutagenesis, the researchers introduced these amino acid changes into envelope proteins made in the laboratory and determined that just two of these changes were responsible for the neutralization sensitivity of the HIV-1 variant. The introduction of these two changes into the neutralization resistant variant and into the unrelated envelope sequences of another subtype A (common in Africa) HIV-1 variant and a subtype B HIV-1 (common in Europe and the Western Hemisphere) variant increased the sensitivity of all these viruses to antibody neutralization.
What Do These Findings Mean?
These findings show that two amino acid changes in gp41 of a neutralization-sensitive HIV-1 variant are responsible for the sensitivity of this variant to several neutralizing antibodies. The finding that the inclusion of these changes in the envelope protein of neutralization-resistant HIV-1 variants greatly increases their sensitivity to neutralizing antibodies indicates that the normally shielded regions of the protein are somehow made accessible to antibody by these changes. One possibility is that the amino acid changes might modify the overall shape of the envelope protein, thus exposing multiple, normally hidden regions in the HIV-1 envelope protein to antibodies. Importantly, these findings open up the possibility that the inclusion of these modifications in envelope-based immunogens might improve the ability of vaccines to generate broadly neutralizing antibodies against HIV-1.
Additional Information.
Please access these Web sites via the online version of this summary at
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIVInSite has comprehensive information on all aspects of HIV/AIDS, including links to resources dealing with HIV vaccine development
Information is available from Avert, an international AIDS charity, on all aspects of HIV and AIDS, including HIV vaccines
The US Centers for Disease Control and prevention provides information on HIV/AIDS including information on its HIV vaccine unit (in English and some information in Spanish)
The AIDS Vaccine Clearinghouse provides clear information about HIV vaccine science, research and product development
The International AIDS Vaccine Initiative also provides straightforward information about the development of HIV vaccines
PMCID: PMC2174964  PMID: 18177204
11.  Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1 
PLoS Pathogens  2014;10(12):e1004552.
To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.
Author Summary
Developing a vaccine against HIV-1 is a priority, but it remains unclear whether immunizations in humans can elicit potent broadly neutralizing antibodies able to prevent HIV-1 transmission. Llamas possess heavy chain only antibodies and conventional heavy and light chain antibodies. We previously reported the heavy chain only antibody J3, which potently neutralizes more than 95% of HIV strains, and was induced by immunization. Here we immunized two further llamas and elicited three novel broadly neutralizing heavy chain only antibodies, which were identified by high-throughput screening. These neutralizing llama antibodies target different areas of the CD4-binding site of the virus, therefore breadth and potency are increased when they are used in combination. To gain greater understanding of how the llama immunizations worked, deep sequencing of the HIV binding region of the antibodies was performed. This revealed that the antibodies were matured fully only in response to the protein immunogens. Furthermore, the VHH elicited in different animals, while sharing functional hallmarks, were encoded by distinct sequences and thus could not have been identified by a deep sequencing analysis alone. Our results show that immunization can potentially induce protective antibodies in llamas and provide a method to more extensively evaluate immunization studies.
PMCID: PMC4270772  PMID: 25522326
12.  Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China 
PLoS Medicine  2006;3(11):e443.
Dating back to the first epidemic among injection drug users in 1989, the Yunnan province has had the highest number of human immunodeficiency virus type 1 (HIV-1) infections in China. However, the molecular epidemiology of HIV-1 in Yunnan has not been fully characterized.
Methods and Findings
Using immunoassays, we identified 103,015 accumulated cases of HIV-1 infections in Yunnan between 1989 and 2004. We studied 321 patients representing Yunnan's 16 prefectures from four risk groups, 11 ethnic populations, and ten occupations. We identified three major circulating subtypes: C/CRF07_BC/CRF08_BC (53%), CRF01_AE (40.5%), and B (6.5%) by analyzing the sequence of p17, which is part of the gag gene. For patients with known risk factors, 90.9% of injection drug users had C/CRF07_BC/CRF08_BC viruses, whereas 85.4% of CRF01_AE infections were acquired through sexual transmission. No distinct segregation of CRF01_AE viruses was found among the Dai ethnic group. Geographically, C/CRF07_BC/CRF08_BC was found throughout the province, while CRF01_AE was largely confined to the prefectures bordering Myanmar. Furthermore, C/CRF07_BC/CRF08_BC viruses were found to consist of a group of viruses, including C, CRF08_BC, CRF07_BC, and new BC recombinants, based on the characterization of their reverse transcriptase genes.
This is the first report of a province-wide HIV-1 molecular epidemiological study in Yunnan. While C/CRF07_BC/CRF08_BC and CRF01_AE are codominant, the discovery of many sexually transmitted CRF01_AE cases is new and suggests that this subtype may lead to a new epidemic in the general Chinese population. We discuss implications of our results for understanding the evolution of the HIV-1 pandemic and for vaccine development.
This is a molecular epidemiology study of circulating HIV strains and subtypes in Yunnan province, which has China's largest number of HIV-infected individuals.
Editors' Summary
The first human immunodeficiency virus (HIV) cases in China were seen in 1989 in Yunnan, a region of south-western China. This area borders Myanmar, Laos, and Vietnam, and is a major entry point for illegal drugs into China. The initial HIV outbreak in this area was in injecting drug users, but HIV is beginning to affect other groups of people in the Yunnan province and is becoming more common across China. There is still not much known about the different types of HIV virus in China and which parts of the population are most likely to be infected. This knowledge is important because it can help people to understand how the epidemic started and how it is likely to spread in the future, and because it helps direct efforts for HIV education and prevention. It is also necessary for the future design of appropriate HIV vaccines.
Why Was This Study Done?
The Yunnan province has the highest rate of HIV-infected individuals in China. It is an important entry point of new HIV virus types into China, and some of the HIV types found in patients in other parts of China appear to have spread from Yunnan. A group of researchers from the United States and China wanted to look at the different types of HIV virus that were infecting people in the Yunnan province and to work out how these types had evolved over the course of the HIV epidemic in China. They focused on human immunodeficiency virus type 1 (HIV-1), the most common form of HIV virus worldwide and also the most infectious. There are at least nine distinct subtypes of HIV-1, and the virus continues to evolve and to form new subtypes.
What Did the Researchers Do and Find?
They collected blood samples from 321 HIV-infected individuals who represented a broad cross-section of the population of Yunnan (people from all geographic parts of the province, 11 ethnic populations, different occupations, etc.) and analyzed the genetic information of the viruses found in these blood samples. Because HIV evolves very rapidly, the genetic information differs between different virus subtypes, and the researchers could therefore tell which subtypes were infecting which subsets of the population. The researchers identified three distinct subtypes of HIV-1: “B” (in about 6.5% of the samples), a group of “C” variants (C/CRF07_BC/CRF08_BC in 53% of the samples), and CRF01_AE (in 40.5%). The CRF01_AE subtype had not previously been reported at such high levels in the Chinese population, and people who were thought to have been infected with HIV through sexual contact (as opposed to contaminated needles) were more likely to be infected with that particular subtype.
What Do These Findings Mean?
The results show a dynamic and evolving pattern of HIV types in the Yunnan province, segregating among different parts of the population. Sexual transmission appears to be on the rise, suggesting that the epidemic could spread rapidly from high-risk groups such as drug users to the general population. HIV/acquired immunodeficiency syndrome (AIDS) education and prevention efforts in the general population are therefore urgently needed. It is also likely that some of the developments of the HIV epidemic in the Yunnan province will be similar in other parts of China as the various subtypes spread. The results of the study also have implications for future HIV vaccine development. Given the range of subtypes, it will be necessary either to develop vaccines that can protect against all the circulating subtypes, or to have a cocktail of several vaccines that each protects against some of them.
Additional Information.
Please access these Web sites via the online version of this summary at
Information from AVERT, an international AIDS charity on HIV subtypes and HIV in China
The UNAIDS on AIDS in Asia
The China AIDS Network—a charity devoted to AIDS research in China
PMCID: PMC1635743  PMID: 17105339
13.  Dissecting the Neutralizing Antibody Specificities of Broadly Neutralizing Sera from Human Immunodeficiency Virus Type 1-Infected Donors▿  
Journal of Virology  2007;81(12):6548-6562.
Attempts to elicit broadly neutralizing antibody responses by human immunodeficiency virus type 1 (HIV-1) vaccine antigens have been met with limited success. To better understand the requirements for cross-neutralization of HIV-1, we have characterized the neutralizing antibody specificities present in the sera of three asymptomatic individuals exhibiting broad neutralization. Two individuals were infected with clade B viruses and the third with a clade A virus. The broadly neutralizing activity could be exclusively assigned to the protein A-reactive immunoglobulin G (IgG) fraction of all three donor sera. Neutralization inhibition assays performed with a panel of linear peptides corresponding to the third hypervariable (V3) loop of gp120 failed to inhibit serum neutralization of a panel of HIV-1 viruses. The sera also failed to neutralize chimeric simian immunodeficiency virus (SIV) and HIV-2 viruses displaying highly conserved gp41-neutralizing epitopes, suggesting that antibodies directed against these epitopes likely do not account for the broad neutralizing activity observed. Polyclonal IgG was fractionated on recombinant monomeric clade B gp120, and the neutralization capacities of the gp120-depleted samples were compared to that of the original polyclonal IgG. We found that the gp120-binding antibody population mediated neutralization of some isolates, but not all. Overall, the data suggest that broad neutralization results from more than one specificity in the sera but that the number of these specificities is likely small. The most likely epitope recognized by the monomeric gp120 binding neutralizing fraction is the CD4 binding site, although other epitopes, such as the glycan shield, cannot be excluded.
PMCID: PMC1900098  PMID: 17409160
14.  The Breadth and Titer of Maternal HIV-1-Specific Heterologous Neutralizing Antibodies Are Not Associated with a Lower Rate of Mother-to-Child Transmission of HIV-1 
Journal of Virology  2012;86(19):10540-10546.
It has been hypothesized that neutralizing antibodies (NAbs) should have broad specificity to be effective in protection against diverse HIV-1 variants. The mother-to-child transmission model of HIV-1 provides the opportunity to examine whether the breadth of maternal NAbs is associated with protection of infants from infection. Samples were obtained at delivery from 57 transmitting mothers (T) matched with 57 nontransmitting mothers (NT) enrolled in the multicenter French perinatal cohort (ANRS EPF CO1) between 1990 and 1996. Sixty-eight (59.6%) and 46 (40.4%) women were infected by B and non-B viruses, respectively. Neutralization assays were carried out with TZM-bl cells, using a panel of 10 primary isolates of 6 clades (A, B, C, F, CRF01_AE, and CRF02_AG), selected for their moderate or low sensitivity to neutralization. Neutralization breadths were not statistically different between T and NT mothers. However, a few statistically significant differences were observed, with higher frequencies or titers of NAbs toward several individual strains for NT mothers when the clade B-infected or non-clade B-infected mothers were analyzed separately. Our study confirms that the breadth of maternal NAbs is not associated with protection of infants from infection.
PMCID: PMC3457297  PMID: 22811522
15.  Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates 
PLoS ONE  2011;6(8):e23233.
Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.
PMCID: PMC3150420  PMID: 21829720
16.  Rapid Escape from Preserved Cross-Reactive Neutralizing Humoral Immunity without Loss of Viral Fitness in HIV-1-Infected Progressors and Long-Term Nonprogressors▿  
Journal of Virology  2010;84(7):3576-3585.
A substantial proportion of human immunodeficiency virus type 1 (HIV-1)-infected individuals has cross-reactive neutralizing activity in serum, with a similar prevalence in progressors and long-term nonprogressors (LTNP). We studied whether disease progression in the face of cross-reactive neutralizing serum activity is due to fading neutralizing humoral immunity over time or to viral escape. In three LTNP and three progressors, high-titer cross-reactive HIV-1-specific neutralizing activity in serum against a multiclade pseudovirus panel was preserved during the entire clinical course of infection, even after AIDS diagnosis in progressors. However, while early HIV-1 variants from all six individuals could be neutralized by autologous serum, the autologous neutralizing activity declined during chronic infection. This could be attributed to viral escape and the apparent inability of the host to elicit neutralizing antibodies to the newly emerging viral escape variants. Escape from autologous neutralizing activity was not associated with a reduction in the viral replication rate in vitro. Escape from autologous serum with cross-reactive neutralizing activity coincided with an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites in the viral envelope. Positive selection pressure was observed in the variable regions in envelope, suggesting that, at least in these individuals, these regions are targeted by humoral immunity with cross-reactive potential. Our results may imply that the ability of HIV-1 to rapidly escape cross-reactive autologous neutralizing antibody responses without the loss of viral fitness is the underlying explanation for the absent effect of potent cross-reactive neutralizing humoral immunity on the clinical course of infection.
PMCID: PMC2838121  PMID: 20071586
17.  Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines 
Journal of Virology  2005;79(10):6089-6101.
A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.
PMCID: PMC1091694  PMID: 15857994
18.  Identification of broadly neutralizing antibody epitopes in the HIV-1 envelope glycoprotein using evolutionary models 
Virology Journal  2013;10:347.
Identification of the epitopes targeted by antibodies that can neutralize diverse HIV-1 strains can provide important clues for the design of a preventative vaccine.
We have developed a computational approach that can identify key amino acids within the HIV-1 envelope glycoprotein that influence sensitivity to broadly cross-neutralizing antibodies. Given a sequence alignment and neutralization titers for a panel of viruses, the method works by fitting a phylogenetic model that allows the amino acid frequencies at each site to depend on neutralization sensitivities. Sites at which viral evolution influences neutralization sensitivity were identified using Bayes factors (BFs) to compare the fit of this model to that of a null model in which sequences evolved independently of antibody sensitivity. Conformational epitopes were identified with a Metropolis algorithm that searched for a cluster of sites with large Bayes factors on the tertiary structure of the viral envelope.
We applied our method to ID50 neutralization data generated from seven HIV-1 subtype C serum samples with neutralization breadth that had been tested against a multi-clade panel of 225 pseudoviruses for which envelope sequences were also available. For each sample, between two and four sites were identified that were strongly associated with neutralization sensitivity (2ln(BF) > 6), a subset of which were experimentally confirmed using site-directed mutagenesis.
Our results provide strong support for the use of evolutionary models applied to cross-sectional viral neutralization data to identify the epitopes of serum antibodies that confer neutralization breadth.
PMCID: PMC4220805  PMID: 24295501
HIV; Antibodies; Neutralization sensitivity; Epitope prediction; Evolutionary model
19.  Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies ▿  
Journal of Virology  2009;84(3):1439-1452.
The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1+) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1+ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.
PMCID: PMC2812321  PMID: 19939925
20.  Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits 
Journal of Virology  2014;88(22):12949-12967.
Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens.
IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating potent neutralization are likely to be critical constituents in an effective HIV vaccine. However, vaccines tested thus far have elicited only weak antibody responses and very modest, waning protection. We hypothesized that B cells develop broad antibodies by exposure to the evolving viral envelope population and tested this concept using multiple envelopes from two subjects who developed neutralization breadth within a few years of infection. We compared different combinations of envelopes from each subject to identify the most effective immunogens and regimens. In each subject, use of HIV envelopes circulating during the early development and maturation of breadth generated more-potent antibodies that were modestly cross neutralizing. These data suggest a new approach to identifying envelope immunogens that may be more effective in generating protective antibodies in humans.
PMCID: PMC4249069  PMID: 25210191
21.  The Ability of an Oligomeric Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Antigen To Elicit Neutralizing Antibodies against Primary HIV-1 Isolates Is Improved following Partial Deletion of the Second Hypervariable Region 
Journal of Virology  2001;75(12):5526-5540.
Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162ΔV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840–7845, 1998). This observation led us to propose that the modified, SF162ΔV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162ΔV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162ΔV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.
PMCID: PMC114265  PMID: 11356960
22.  Selected HIV-1 Env Trimeric Formulations Act as Potent Immunogens in a Rabbit Vaccination Model 
PLoS ONE  2013;8(9):e74552.
Ten to 30% of HIV-1 infected subjects develop broadly neutralizing antibodies (bNAbs) during chronic infection. We hypothesized that immunizing rabbits with viral envelope glycoproteins (Envs) from these patients may induce bNAbs, when formulated as a trimeric protein and in the presence of an adjuvant.
Based on in vitro neutralizing activity in serum, patients with bNAbs were selected for cloning of their HIV-1 Env. Seven stable soluble trimeric gp140 proteins were generated from sequences derived from four adults and two children infected with either clade A or B HIV-1. From one of the clade A Envs both the monomeric and trimeric Env were produced for comparison. Rabbits were immunized with soluble gp120 or trimeric gp140 proteins in combination with the adjuvant dimethyl dioctadecyl ammonium/trehalose dibehenate (CAF01). Env binding in rabbit immune serum was determined using ELISAs based on gp120-IIIB protein. Neutralizing activity of IgG purified from rabbit immune sera was measured with the pseudovirus-TZMbl assay and a PBMC-based neutralization assay for selected experiments.
It was initially established that gp140 trimers induce better antibody responses over gp120 monomers and that the adjuvant CAF01 was necessary for such strong responses. Gp140 trimers, based on HIV-1 variants from patients with bNAbs, were able to elicit both gp120IIIB specific IgG and NAbs to Tier 1 viruses of different subtypes. Potency of NAbs closely correlated with titers, and an gp120-binding IgG titer above a threshold of 100,000 was predictive of neutralization capability. Finally, peptide inhibition experiments showed that a large fraction of the neutralizing IgG was directed against the gp120 V3 region.
Our results indicate that the strategy of reverse immunology based on selected Env sequences is promising when immunogens are delivered as stabilized trimers in CAF01 adjuvant and that the rabbit is a valuable model for HIV vaccine studies.
PMCID: PMC3759472  PMID: 24023951
23.  Development of a Poliovirus Neutralization Test with Poliovirus Pseudovirus for Measurement of Neutralizing Antibody Titer in Human Serum ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(11):1889-1894.
In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV.
PMCID: PMC3209023  PMID: 21880850
24.  Broad HIV-1 Neutralizing Antibody Response Induced by Heterologous Gp140/Gp145 DNA Prime-Vaccinia Boost Immunization 
Vaccine  2012;30(28):4135-4143.
To develop an effective HIV vaccine strategy that can induce cross-reactive neutralizing antibody.
Codon-optimized gp140 and gp145 env genes derived from HIV-1cn54, a CRF07 B′/C recombinant strain, were constructed as DNA and recombinant Tiantan vaccinia (rTV) vaccines. The effect of heterologous immunization with gp140 and gp145 was tested in mice and guinea pigs. T cell responses were detected using the IFN-γ ELISPOT assay. A panel of primary isolates of clade B′ and B′/C HIV-1 and TZM-bl cells was used to determine the neutralizing activity of immunized sera.
The neutralizing antibodies (NAbs) induced by the heterologous immunogen immunization neutralized all HIV-1 B′ and B′/C primary isolates in the guinea pig model. Gp145 and gp140 heterologous prime-boost induced the best neutralizing antibody response with a broad neutralizing spectrum and the highest titer of 1:270 at 6 weeks after the last inoculation. However, the T cell response to HIV-1 peptides was significantly weaker than the gp145+gp145 homologous prime-boost.
This heterologous prime-boost immunization strategy could be used to design immunogen-generating broad neutralizing antibodies against genetic variance pathogens.
PMCID: PMC3422682  PMID: 22561314
HIV; vaccine; neutralizing antibody; heterologous prime-boost immunization
25.  Breadth of Neutralizing Antibodies Elicited by Stable, Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs ▿ †  
Journal of Virology  2010;84(7):3270-3279.
The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.
PMCID: PMC2838122  PMID: 20053749

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