Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.

Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice

Human immunodeficiency virus type 1 (HIV-1) subtype C is the predominant HIV in southern Africa, and is the target of a number of recent vaccine candidates. It has been proposed that a heterologous prime/boost vaccination strategy may result in stronger, broader and more prolonged immune responses. Since HIV-1 Gag Pr55 polyprotein can assemble into virus-like particles (VLPs) which have been shown to induce a strong cellular immune response in animals, we showed that a typical southern African subtype C Pr55 protein expressed in insect cells via recombinant baculovirus could form VLPs. We then used the baculovirus-produced VLPs as a boost to a subtype C HIV-1 gag DNA prime vaccination in mice. This study shows that a low dose of HIV-1 subtype C Gag VLPs can significantly boost the immune response to a single subtype C gag DNA inoculation in mice. These results suggest a possible vaccination regimen for humans. © 2004 SGM.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

Determinants of hepatitis A vaccine immunity in a cohort of human immunodeficiency virus-infected children living in Switzerland

Vaccination in HIV-infected children is often less effective than in healthy children. The goal of this study was to assess vaccine responses to hepatitis A virus (HAV) in HIV-infected children. Children of the Swiss Mother and Child HIV Cohort Study (MoCHiV) were enrolled prospectively. Recommendations for initial, catch-up, and additional HAV immunizations were based upon baseline antibody concentrations and vaccine history. HAV IgG was assessed by enzyme-linked immunosorbent assay (ELISA) with a protective cutoff value defined as ≥10 mIU/ml. Eighty-seven patients were included (median age, 11 years; range, 3.4 to 21.2 years). Forty-two patients were seropositive (48.3%) for HAV. Among 45 (51.7%) seronegative patients, 36 had not received any HAV vaccine dose and were considered naïve. Vaccine responses were assessed after the first dose in 29/35 naïve patients and after the second dose in 33/39 children (25 initially naïve patients, 4 seronegative patients, and 4 seropositive patients that had already received 1 dose of vaccine). Seroconversion was 86% after 1 dose and 97% after 2 doses, with a geometric mean concentration of 962 mIU/ml after the second dose. A baseline CD4(+) T cell count below 750 cells/μl significantly reduced the post-2nd-dose response (P = 0.005). Despite a high rate of seroconversion, patients with CD4(+) T cell counts of <750/μl had lower anti-HAV antibody concentrations. This may translate into a shorter protection time. Hence, monitoring humoral immunity may be necessary to provide supplementary doses as needed.

Determinants of vaccine immunity in the cohort of human immunodeficiency virus-infected children living in Switzerland

BACKGROUND: Human immunodeficiency virus (HIV)-infected children are at increased risk of infections caused by vaccine preventable pathogens, and specific immunization recommendations have been issued. METHODS: A prospective national multicenter study assessed how these recommendations are followed in Switzerland and how immunization history correlates with vaccine immunity. RESULTS: Among 87 HIV-infected children (mean age: 11.1 years) followed in the 5 Swiss university hospitals and 1 regional hospital, most (76%) had CD4 T cells >25%, were receiving highly active antiretroviral treatment (79%) and had undetectable viral load (60%). Immunization coverage was lower than in the general population and many lacked serum antibodies to vaccine-preventable pathogens, including measles (54%), varicella (39%), and hepatitis B (65%). The presence of vaccine antibodies correlated most significantly with having an up-to-date immunization history (P<0.05). An up-to-date immunization history was not related to age, immunologic stage, or viremia but to the referral medical center. CONCLUSIONS: All pediatricians in charge of HIV-infected children are urged to identify missing immunizations in this high-risk population.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+ T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Equal IgG antibody response to pneumococcal vaccination in all stages of human immunodeficiency virus disease.

To evaluate the immunogenicity and safety of a 23-valent pneumococcal vaccine in human immunodeficiency virus (HIV)-seropositive patients, 80 men and 18 women received 1 dose of the vaccine (Pneumo 23; Pasteur Mérieux MSD, Brussels). The total IgG antibody response against all 23 Streptococcus pneumoniae capsular antigens was measured. Antibody levels were expressed in arbitrary units per microliter, referring to a standard curve. Geometric mean titers of the total IgG capsular antibodies on the day of vaccination and 30-45 days later were compared. The ratios of titers after and before vaccination in patients with > 500, 200-500, and < 200 CD4 lymphocytes/microL were 10, 10, and 12.6, respectively. Nonresponse (ratio < 4) occurred in 17% of patients and was unrelated to CD4 cell count. The vaccine was well tolerated; no serious side effects occurred. In 83% of the patients with HIV infection, the total antipneumococcal IgG level was higher after vaccination.

Neutralization activity in a geographically diverse East London cohort of human immunodeficiency virus type 1-infected patients: clade C infection results in a stronger and broader humoral immune response than clade B infection.

The array of human immunodeficiency virus (HIV) subtypes encountered in East London, an area long associated with migration, is unusually heterogeneous, reflecting the diverse geographical origins of the population. In this study it was shown that viral subtypes or clades infecting a sample of HIV type 1 (HIV-1)-positive individuals in East London reflect the global pandemic. The authors studied the humoral response in 210 treatment-naïve chronically HIV-1-infected (>1 year) adult subjects against a panel of 12 viruses from six different clades. Plasmas from individuals infected with clade C, but also plasmas from clade A, and to a lesser degree clade CRF02_AG and CRF01_AE, were significantly more potent at neutralizing the tested viruses compared with plasmas from individuals infected with clade B. The difference in humoral robustness between clade C- and B-infected patients was confirmed in titration studies with an extended panel of clade B and C viruses. These results support the approach to develop an HIV-1 vaccine that includes clade C or A envelope protein (Env) immunogens for the induction of a potent neutralizing humoral response.

Detection of immunoglobulin G antibodies to Neospora caninum in humans: High seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders

Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.

Conditional reduction of human immunodeficiency virus type 1 replication by a gain-of-herpes simplex virus 1 thymidine kinase function.

The development of an effective vaccine for human immunodeficiency virus type 1 (HIV-1) would be a major advance toward controlling the AIDS pandemic. Several disparate strategies for a safe and effective HIV vaccine have been proposed. Recent data suggest that loss-of-function live-attenuated virus could be a safe lentivirus vaccine. Here, we propose a gain-of-function approach that can complement loss-of-function in enhancing the safety profile of a live-attenuated virus. We describe an example in which ganciclovir (GCV) was used to treat effectively nef(-)HIV-1 engineered to express herpes simplex virus (HSV-1) thymidine kinase (TK). This treatment was found to be highly efficient in controlling HIV-1 spread in tissue culture and in a small animal (hu-PBL-SCID) model. We demonstrate that one distinct advantage of GCV-HSV-TK treatment is the elimination of integrated proviruses, a goal not easily achieved with other antiretrovirals.

Sleep electroencephalogram delta-frequency amplitude, night plasma levels of tumor necrosis factor alpha, and human immunodeficiency virus infection.

We tested the hypothesis that increases in tumor necrosis factor alpha (TNF-alpha) induced by human immunodeficiency virus (HIV) are associated with the increases in slow-wave sleep seen in early HIV infection and the decrease with sleep fragmentation seen in advanced HIV infection. Nocturnal sleep disturbances and associated fatigue contribute to the disability of HIV infection. TNF-alpha causes fatigue in clinical use and promotes slow-wave sleep in animal models. With slow progress toward a vaccine and weak effects from current therapies, efforts are directed toward extending productive life of HIV-infected individuals and shortening the duration of disability in terminal illness. We describe previously unrecognized nocturnal cyclic variations in plasma levels of TNF-alpha in all subjects. In 6 of 10 subjects (1 control subject, 3 HIV-seropositive patients with CD4+ cell number > 400 cells per microliters, and 2 HIV-positive patients with CD4+ cell number < 400 cells per microliters), these fluctuations in TNF-alpha were coupled to the known rhythm of electroencephalogram delta amplitude (square root of power) during sleep. This coupling was not present in 3 HIV-positive subjects with CD4+ cell number < 400 cells per microliters and 1 control subject. In 5 HIV subjects with abnormally low CD4+ cell counts ( < 400 cells per microliters), the number of days since seroconversion correlated significantly with low correlation between TNF-alpha and delta amplitude. We conclude that a previously unrecognized normal, physiological coupling exists between TNF-alpha and delta amplitude during sleep and that the lessened likelihood of this coupling in progressive HIV infection may be important in understanding fatigue-related symptoms and disabilities.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.