Recent advances and future perspectives on carbohydrate-based cancer vaccines and therapeutics.

Carbohydrates are abundantly expressed on the surface of both eukaryotic and prokaryotic cells, often as post translational modifications of proteins. Glycoproteins are recognized by the immune system and can trigger both innate and humoral responses. This feature has been harnessed to generate vaccines against polysaccharide-encapsulated bacteria such as Streptococcus pneumoniae, Hemophilus influenzae type b and Neisseria meningitidis. In cancer, glycosylation plays a pivotal role in malignancy development and progression. Since glycans are specifically expressed on the surface of tumor cells, they have been targeted for the discovery of anticancer preventive and therapeutic treatments, such as vaccines and monoclonal antibodies. Despite the various efforts made over the last years, resulting in a series of clinical studies, attempts of vaccination with carbohydrate-based candidates have proven unsuccessful, primarily due to the immune tolerance often associated with these glycans. New strategies are thus deployed to enhance carbohydrate-based cancer vaccines. Moreover, lessons learned from glycan immunobiology paved the way to the development of new monoclonal antibodies specifically designed to recognize cancer-bound carbohydrates and induce tumor cell killing. Herein we provide an overview of the immunological principles behind the immune response towards glycans and glycoconjugates and the approaches exploited at both preclinical and clinical level to target cancer-associated glycans for the development of vaccines and therapeutic monoclonal antibodies. We also discuss gaps and opportunities to successfully advance glycan-directed cancer therapies, which could provide patients with innovative and effective treatments.

Vaccinology in the post-COVID-19 era.

The COVID-19 pandemic is a shocking reminder of how our world would look in the absence of vaccination. Fortunately, new technologies, the pace of understanding new and existing pathogens, and the increased knowledge of the immune system allow us today to develop vaccines at an unprecedented speed. Some of the vaccine technologies that are fast-tracked by the urgency of COVID-19 may also be the answer for other health priorities, such as antimicrobial resistance, chronic infections, and cancer, that the post-COVID-19 world will urgently need to face. This perspective analyzes the way COVID-19 is transforming vaccinology and the opportunities for vaccines to have an increasingly important role in health and well-being.

Late boosting of the RV144 regimen with AIDSVAX B/E and ALVAC-HIV in HIV-uninfected Thai volunteers: a double-blind, randomised controlled trial.

BACKGROUND: The RV144 phase 3 vaccine trial in Thailand demonstrated that ALVAC-HIV (vCP1521) and AIDSVAX B/E administration over 6 months resulted in a 31% efficacy in preventing HIV acquisition. In this trial, we assessed the immunological effect of an additional vaccine boost to the RV144 regimen at varying intervals between the priming vaccine series and the boost. METHODS: RV306 is a double-blind, placebo-controlled, randomised clinical trial done at three clinical sites in Thailand. Eligible volunteers were HIV-uninfected individuals aged 20-40 years who were at low risk for HIV infection and in good health. A randomisation schedule was centrally generated with fixed sized strata for Research Institute for Health Sciences Chiang Mai and combined Bangkok clinics. Participants were randomly assigned to one of five groups and then further randomly assigned to either vaccine or placebo. All participants received the primary RV144 vaccine series at months 0, 1, 3, and 6. Group 1 received no additional boost, group 2 received additional AIDSVAX B/E and ALVAC-HIV (vCP1521) or placebo at month 12, group 3 received AIDSVAX B/E alone or placebo at month 12, group 4a received AIDSVAX B/E and ALVAC-HIV or placebo at month 15, and group 4b received AIDSVAX B/E and ALVAC-HIV or placebo at month 18. Primary outcomes were safety and tolerability of these vaccination regimens and cellular and humoral immune responses compared between the RV144 series alone and regimens with late boosts at different timepoints. Safety and tolerability outcomes were assessed by evaluating local and systemic reactogenicity and adverse events in all participants. This trial is registered at ClinicalTrials.gov (NCT01931358); clinical follow-up is now complete. FINDINGS: Between Oct 28, 2013, and April 29, 2014, 367 participants were enrolled, of whom 27 were assigned active vaccination in group 1, 102 in group 2, 101 in group 3, 52 in group 4a, 51 in group 4b, and 34 combined placebo across all the groups. No vaccine-related serious adverse events were recorded. Occurrence and severity of local and systemic reactogenicity were similar across active groups. Groups with late boosts (groups 2, 3, 4a, and 4b) had increased peak plasma IgG-binding antibody levels against gp70 V1V2 relative to group 1 vaccine recipients with no late boost (gp70 V1V2 92TH023 adjusted p<0·02 for each; gp70 V1V2 CaseA2 adjusted p<0·0001 for each). Boosting at month 12 (groups 2 and 3) did not increase gp120 responses compared with the peak responses after the RV144 priming regimen at month 6; however, boosting at month 15 (group 4a) improved responses to gp120 A244gD- D11 (p=0·0003), and boosting at month 18 (group 4b) improved responses to both gp120 A244gD- D11 (p<0·0001) and gp120 MNgD- D11 (p=0·0016). Plasma IgG responses were significantly lower among vaccine recipients boosted at month 12 (pooled groups 2 + 3) than at month 15 (group 4a; adjusted p<0·0001 for each, except for gp70 V1V2 CaseA2, p=0·0142) and at month 18 (group 4b; all adjusted p<0·001). Boosting at month 18 versus month 15 resulted in a significantly higher plasma IgG response to gp120 antigens (all adjusted p<0·01) but not gp70 V1V2 antigens. CD4 functionality and polyfunctionality scores after stimulation with HIV-1 Env peptides (92TH023) increased with delayed boosting. Groups with late boosts had increased functionality and polyfunctionality scores relative to vaccine recipients with no late boost (all adjusted p<0·05, except for the polyfunctionality score in group 1 vs group 4b, p<0·01). INTERPRETATION: Taken together, these results suggest that additional boosting of the RV144 regimen with longer intervals between the primary vaccination series and late boost improved immune responses and might improve the efficacy of preventing HIV acquisition. FUNDING: US National Institute of Allergy and Infectious Diseases and US Department of the Army.

Immune correlates of the Thai RV144 HIV vaccine regimen in South Africa.

One of the most successful HIV vaccines to date, the RV144 vaccine tested in Thailand, demonstrated correlates of protection including cross-clade V1V2 immunoglobulin G (IgG) breadth, Env-specific CD4+ T cell polyfunctionality, and antibody-dependent cellular cytotoxicity (ADCC) in vaccinees with low IgA binding. The HIV Vaccine Trials Network (HVTN) 097 trial evaluated this vaccine regimen in South Africa, where clade C HIV-1 predominates. We compared cellular and humoral responses at peak and durability immunogenicity time points in HVTN 097 and RV144 vaccinee samples, and evaluated vaccine-matched and cross-clade immune responses. At peak immunogenicity, HVTN 097 vaccinees exhibited significantly higher cellular and humoral immune responses than RV144 vaccinees. CD4+ T cell responses were more frequent in HVTN 097 irrespective of age and sex, and CD4+ T cell Env-specific functionality scores were higher in HVTN 097. Env-specific CD40L+ CD4+ T cells were more common in HVTN 097, with individuals having this pattern of expression demonstrating higher median antibody responses to HIV-1 Env. IgG and IgG3 binding antibody rates and response magnitude to gp120 vaccine- and V1V2 vaccine-matched antigens were higher or comparable in HVTN 097 than in RV144 ADCC, and ADCP functional antibody responses were elicited in HVTN 097. Env-specific IgG and CD4+ Env responses declined significantly over time in both trials. Overall, cross-clade immune responses associated with protection were better than expected in South Africa, suggesting wider applicability of this regimen.

Application of replication-defective West Nile virus vector to non-flavivirus vaccine targets.

The RepliVax vaccine platform(RV) is based on flavivirus genomes that are rationally attenuated by deletion. The self-limiting infection provided by RV has been demonstrated to be safe, highly immunogenic and efficacious for several vaccine candidates against flaviviruses. Here respiratory syncytial virus (RSV) F, influenza virus HA, and simian immunodeficiency virus (SIV) Env proteins were expressed in place of either prM-E or C-prM-E gene deletions of the West Nile (WN) virus genome. The resulting RV-RSV, -influenza and -SIV vaccine prototypes replicated efficiently in complementing helper cells expressing the WN structural proteins in trans. Expressed antigens exhibited correct post-translational processing and the RV recombinants were shown to be highly attenuated and immunogenic in mice, eliciting strong antigen-specific antibodies as well as detectable T-cell responses. These data support the utility of RV vectors for development of vaccines against non-flavivirus targets including rabies and HIV.

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates.

The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia virus B19 protein, an inhibitor of the type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited high levels of T cell and humoral immune responses, with the vector containing a deletion in B19R inducing a trend toward a higher magnitude of CD4+ and CD8+ T cell responses and neutralization of some HIV-1 strains. These poxvirus vectors could be considered HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Adjuvant-dependent innate and adaptive immune signatures of risk of SIVmac251 acquisition.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates.

UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

The stem of vesicular stomatitis virus G can be replaced with the HIV-1 Env membrane-proximal external region without loss of G function or membrane-proximal external region antigenic properties.

The structure of the HIV-1 envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently, although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the preattachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G-stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.

The canarypox virus vector ALVAC induces distinct cytokine responses compared to the vaccinia virus-based vectors MVA and NYVAC in rhesus monkeys.

Despite the growing use of poxvirus vectors as vaccine candidates for multiple pathogens and cancers, their innate stimulatory properties remain poorly characterized. Here we show that the canarypox virus-based vector ALVAC induced distinct systemic proinflammatory and antiviral cytokine and chemokine levels following the vaccination of rhesus monkeys compared to the vaccinia virus-based vectors MVA and NYVAC. These data suggest that there are substantial biological differences among leading poxvirus vaccine vectors that may influence resultant adaptive immune responses following vaccination.

Broad neutralization coverage of HIV by multiple highly potent antibodies.

Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.

HIV/AIDS: vaccines and alternate strategies for treatment and prevention.

The symposium "HIV/AIDS: Vaccines and Alternate Strategies for Treatment and Prevention" brought together HIV vaccine researchers to discuss the latest developments in the field. From basic discoveries in virus diversity and mechanisms of neutralization by antibodies to nonhuman primate research and clinical trials of vaccine candidates in volunteers, scientists are making great strides in understanding the mechanisms that may protect against HIV and pathways to achieve this protection through vaccination.

Optimization of a dendritic cell-based assay for the in vitro priming of naïve human CD4+ T cells.

Methods to prime human CD4(+) T cells in vitro would be of significant value for the pre-clinical evaluation of vaccine candidates and other immunotherapeutics. However, to date, there is no reliable method for the induction of primary human T cell responses in the laboratory. Here, we optimized a culture strategy incorporating highly purified lymphocytes and dendritic cells, in the absence of any exogenous growth factors, for the in vitro sensitization of naïve CD4(+) T cells against a variety of protein antigens. This fully autologous approach, which was superior to the more traditional PBMC assay for supporting the induction of primary human T helper cell responses in culture, elicited effector cells capable of producing a variety of Th cytokines, including IFNgamma, TNFalpha, IL-2, IL-5, IL-17 and IL-21, and memory cells that could be restimulated multiple times with a specific antigen. Through simple modifications to this culture method, we evaluated the role of dendritic cell maturation state and regulatory T cells on the sensitization of naïve T helper cells, which highlights its utility for addressing basic questions of human immunobiology. Finally, using the formulated yellow fever vaccine, YF-VAX (R), we provide a proof-of-concept demonstration of the utility of the system for evaluating the T cell immunogenicity of vaccine candidates in a pre-clinical setting.

Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target.

Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1-infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1-infected individuals, primarily infected with non-clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from about 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.

The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus.

The ability to elicit broadly neutralizing antibody responses against HIV-1 is a crucial goal for a prophylactic HIV-1 vaccine. Here, we discuss the difficulties of achieving broad HIV-1 neutralization in the context of both the effective annual human influenza virus vaccine and the need to develop a pandemic influenza vaccine. Immunogen-design strategies are underway to target functionally conserved regions of the HIV-1 envelope glycoproteins, and similar strategies might be applicable to pandemic influenza virus vaccine development. Efforts to develop broadly neutralizing vaccines against either HIV-1 or influenza virus might establish a paradigm for future vaccines against highly variable pathogens.

Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library.

Identification of broadly cross-reactive human monoclonal antibodies (mAbs) has major implications for development of vaccines, inhibitors and research tools. Here we describe a sequential antigen panning (SAP) methodology that may facilitate the selection of such antibodies. An HIV-specific antibody Fab (m18) was selected from a human Fab phage-display library by SAP against several recombinant soluble HIV envelope glycoproteins (Envs) and Env-sCD4 complexes. This Fab bound to a variety of recombinant soluble Envs (gp140s) from primary HIV isolates representing different clades, and inhibited cell fusion and virus entry mediated by Envs of primary HIV isolates. The methodology and the results may have implications for development of HIV vaccines and inhibitors, as well as for identification of antibodies to conserved epitopes on rapidly mutating viruses and cells.

Cell biology of virus entry: a review of selected topics from the 3rd International Frederick meeting.

Following the first two Frederick meetings on virus entry in 1997 [Cell 91 (1997) 721](1) and in 2000 [Cell 101 (2000) 697](2), further developments in our understanding of the multifactorial and multistage process of virus entry, and possible biomedical implications were presented and discussed in a lively fashion by leading scientists from around the world at the third Frederick meeting on the Cell Biology of Viral Entry (May 7-10, Frederick, MD) organized by R. Blumenthal (NCI-Frederick, NIH, Frederick) and E. Hunter (University of Alabama, Birmingham). Unlike the previous two meetings, non-enveloped viruses were not discussed this time, and the focus was how envelope glycoproteins (Envs) mediate entry into cells. Major topics included Env structure, virus receptors, entry intermediates, membrane fusion, fusion kinetics, and rafts. Virus envelope structures will be described in more detail here because the other topics are extensively discussed in the other chapters of this volume.

Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120-CD4-CCR5 complexes.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor, typically CCR5. Here we provide evidence that purified gp120(JR-FL)-CD4-CCR5 complexes exhibit an epitope recognized by a Fab (X5) obtained by selection of a phage display library from a seropositive donor with a relatively high broadly neutralizing serum antibody titer against an immobilized form of the trimolecular complex. X5 bound with high (nM) affinity to a variety of Envs, including primary isolates from different clades and Envs with deleted variable loops (V1, -2, -3). Its binding was significantly increased by CD4 and slightly enhanced by CCR5. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D, E, F, and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore, X5 inhibited cell fusion mediated by Envs from R5, X4, and R5X4 viruses. Of the five broadly cross-reactive HIV-1-neutralizing human monoclonal antibodies known to date, X5 is the only one that exhibits increased binding to gp120 complexed with receptors. These findings suggest that X5 could possibly be used as entry inhibitor alone or in combination with other antiretroviral drugs for the treatment of HIV-1-infected individuals, provide evidence for the existence of conserved receptor-inducible gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-1-infected patients, and have important conceptual and practical implications for the development of vaccines and inhibitors.