Tong-Ming Fu

Universal Influenza B Vaccine Based on the Maturational Cleavage Site of the Hemagglutinin Precursor

ABSTRACT Conventional influenza vaccines can prevent infection, but their efficacy depends on the degree of antigenic “match” between the strains used for vaccine preparation and those circulating in the population. A universal influenza vaccine based on invariant regions of the virus, able to provide broadly cross-reactive protection, without requiring continuous manufacturing update, would solve a major medical need. Since the temporal and geographical dominance of the influenza virus type and/or subtype (A/H3, A/H1, or B) cannot yet be predicted, a universal vaccine, like the vaccines currently in use, should include both type A and type B influenza virus components. However, while encouraging preclinical data are available for influenza A virus, no candidate universal vaccine is available for influenza B virus. We show here that a peptide conjugate vaccine, based on the highly conserved maturational cleavage site of the HA0 precursor of the influenza B virus hemagglutinin, can elicit a protective immune response against lethal challenge with viruses belonging to either one of the representative, non-antigenically cross-reactive influenza B virus lineages. We demonstrate that protection by the HA0 vaccine is mediated by antibodies, probably through effector mechanisms, and that a major part of the protective response targets the most conserved region of HA0, the P1 residue of the scissile bond and the fusion peptide domain. In addition, we present preliminary evidence that the approach can be extended to influenza A virus, although the equivalent HA0 conjugate is not as efficacious as for influenza B virus.

Evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities

Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.

Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

A replication-defective human cytomegalovirus vaccine for prevention of congenital infection

A new replication-defective human cytomegalovirus vaccine shows promise in protecting against this dangerous virus that can cause severe neurodevelopmental defects in babies. Progress toward a human cytomegalovirus vaccine Most adults carry latent human cytomegalovirus, with no apparent symptoms. But if a pregnant woman becomes newly infected or an active infection is triggered, the fetus can be harmed and show severe neurodevelopmental deficits. Wang and colleagues report a key step toward an effective vaccine against this dangerous herpesvirus. They engineered a genetic off switch into the whole virus to render it harmless and show that it can elicit desirable immune responses such as neutralizing antibodies cell-mediated immune responses in several animal species, including nonhuman primates. Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.

Development of HIV-1 Nef vaccine components: immunogenicity study of Nef mutants lacking myristoylation and dileucine motif in mice

In an effort to develop a safe Nef component for use in Cytotoxic T-lymphocyte (CTL)-based HIV-1 vaccines, several versions of Nef constructs lacking myristoylation and dileucine motif were engineered and their abilities to elicit T cell responses were evaluated in mice. Nef-specific murine T cell epitopes were first mapped in three strains of mice (Balb/c, C3H/HeN and C57BL/6), and a pair of dominant Nef-specific CD4(+) and CD8(+) T cell epitopes were identified in C57BL/6 mice. C57BL/6 mice were subsequently immunized with engineered Nef DNA constructs, and Nef-specific CD4(+) and CD8(+) T cell responses were determined. A Nef mutant with simple alanine substitutions at the myristoylation and dileucine sites was impaired in its ability to elicit Nef-specific CD4(+) and CD8(+) T cell responses. Addition of human tissue plasminogen activator (TPA) leader sequence to the N terminus of Nef, which concomitantly inactivates the myristoylation site, significantly enhanced the Nef-specific T cell responses. These findings may have practical implications for developing HIV-1 Nef vaccine component.

Evaluation of Cytotoxic T-Lymphocyte Responses in Human and Nonhuman Primate Subjects Infected with Human Immunodeficiency Virus Type 1 or Simian/Human Immunodeficiency Virus

ABSTRACT Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.

Preclinical evaluations of peptide-conjugate vaccines targeting the antigenic domain-2 of glycoprotein B of human cytomegalovirus

ABSTRACT The Antigenic Domain 2 (AD-2) is a short region near the N-terminus of glycoprotein B of human cytomegalovirus (HCMV). AD-2 has been shown to contain linear epitopes that are targets for neutralizing monoclonal antibodies from human subjects with natural HCMV infection. However, AD-2 appears to be masked by the adjacent immunodominant AD-1 region. We assessed a serum panel from HCMV-seropositive individuals and found a wide range of antibody titers to AD-2; these did not correlate to serum neutralization. To expose potential epitopes in AD-2, we constructed a series of AD-2 peptide-conjugate vaccines. Mice were immunized 3 times and produced high and sustained antibody titers to AD-2 peptides, but neutralization was weak even after a single boost with whole HCMV virions. Rabbits were likewise immunized with AD-2 peptide vaccines, and produced a robust antibody response, but neutralization was inferior to a recombinant gB vaccine with an oil-in-water adjuvant. These results highlight the challenges of developing a peptide-based vaccine specific to the HCMV gB AD-2 region.

Active evolution of memory B-cells specific to viral gH/gL/pUL128/130/131 pentameric complex in healthy subjects with silent human cytomegalovirus infection

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection.Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection.

Functionally inactivated dominant viral antigens of human cytomegalovirus delivered in replication incompetent adenovirus type 6 vectors as vaccine candidates

ABSTRACT T cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

Genome Sequences of Diverse Human Cytomegalovirus Strains with Utility in Drug Screening and Vaccine Evaluation.

ABSTRACT Cytomegalovirus displays genetic heterogeneity, which has implications for antiviral and vaccine development. Many studies have focused on laboratory isolates that have been extensively adapted for growth on fibroblasts. Here, we report whole-genome sequences for 10 human cytomegalovirus (HCMV) strains that readily grow on ARPE-19 human retinal pigment epithelial cells.