Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

A "universal" human influenza A vaccine.

We have previously reported on a universal human influenza A vaccine, based on the external domain of the transmembrane viral M2-protein (M2e) [Nature Medicine 5 (1999) 1119]. M2-protein is scarcely present on the virus but is abundantly expressed on virus-infected cells. The external domain, M2e, is 23-amino acids long and as such weakly immunogenic. But when presented on an appropriate carrier, such as hepatitis B virus core (HBc) particles, it induces a high titer antibody response that in mice effectively protects against a potentially lethal influenza infection. The advantage of M2e as an antigen is the conservation of its sequence that has hardly changed since the first influenza virus was isolated in 1933, despite numerous epidemics and several pandemics. Various constructs, e.g. M2e fused at the N-terminus of the HBc subunit or inserted in the immuno-dominant loop, were evaluated as a vaccine. They conferred full protection when administered together with an adjuvant. Several adjuvants were tested in conjunction with intraperitoneal vaccine administration, while the non-toxic enterotoxin mutant LT(R192G) was used for intranasal vaccination. Appropriate combinations of vaccine construct and adjuvant allowed to obtain anti-M2e IgG2a serum titers above 10,000, and this provided complete protection.

A system for logging incontinence events using a simple disposable sensor.

Many elderly people entering residential or nursing care are already incontinent to some degree, relying on incontinence pads to deal with the consequences. A proportion of these people have been shown to exhibit a regular pattern in their incontinence, which opens up the possibility of mitigating the problem by instituting an individual toileting regime for the person. This can reduce their reliance on incontinence pads, both improving their quality of life, and reducing the cost of care. This paper covers the development and evaluation of a sensor for detecting incontinence events, suitable for use in this setting, and describes the design of an associated electronic logger. The devices form part of an assessment system intended to identify a pattern in incontinence where it exists, and to help with the design of the toilet regime for an individual. The requirement is that the system must reliably record incontinence events, and present the information describing them in a manner appropriate to the users of the devices, who are likely to be non-technical and non-specialist.

A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts.

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to the Plasmodium parasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes of Plasmodium falciparum CS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimal P. falciparum HBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and in Macaca fascicularis (cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4(+)-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in naïve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed with P. falciparum sporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.

Online motion-gated dynamic three-dimensional echocardiography in the fetus--preliminary results.

The aim of this study was to visualise the fetal heart in dynamic three dimensions (4-D) during an ultrasound (US) scan (online), rather than after (offline). With special pairing and sequential setting to minimise interference between two scanners, umbilical arterial Doppler waveforms (UADWs) from one scanner were used as an online motion gating source to trigger simultaneous 3-D cardiac structural data acquisition by another. Of 25 data sets from 10 fetuses, 18 were acquired in 15 to 30 s per set with > or = 50% Doppler waveforms efficiently converted to triggering signals. Of 15 valid 4-D data sets, 10 were reconstructed in 2 to 20 min, compared to over 2 h previously reported (mainly for offline gating). Fine structures (including chordae tendinae and trabecular muscles) were depicted in six sets. The main problems in degrading 4-D images were extensive shadowing (6) from bony structures during rigid mechanical scanning, and random motion artefacts (6) from prolonged setting-up time with a complex combination of several systems. Integration of these systems is, therefore, recommended.

Conversion of umbilical arterial Doppler waveforms to cardiac cycle triggering signals: a preparatory study for online motion-gated three-dimensional fetal echocardiography.

To remove motion artefacts, a device was built to convert "noisy" umbilical arterial Doppler waveforms (UADWs) from an ultrasound (US) system into sharp ECG R-wave-like cardiac cycle triggering signals (CCTSs). These CCTSs were then used to gate a simultaneous (online) 3-D acquisition of sectional fetal echocardiograms from another US system. To test the conversion performance, a study was carried out in sheep fetal twins. Pulmonary arterial flow waveforms (PAFWs) from implanted probes were traced, in the meantime, to determine the reference cardiac cycle. Interference caused by running the two nonsynchronised US systems was controlled to three degrees (not-noticeable, moderate, and severe), together with high (> or = 40 cm/s) and low (< 40) flow velocities on UADWs. The conversion efficiency, assessed by the percentage of UADWs converted into CCTSs, was in the range of 83% to 100% for not-noticeable and moderate interference, and 0% to 71% for severe interference. The triggering accuracy, assessed by [(time lag mean between the onsets of PAFWs and corresponding CCTSs) -- (its 99% confidence level)] / the mean, was 90% to 96% for the not-noticeable interference high- and low-flow groups and for the moderate interference high-flow group; 19% to 93% for the moderate interference low-flow group; and from not obtainable up to 90% for the severe interference groups. The results show that UADWs can be used as a satisfactory online motion-gating source even in the presence of moderate interference. The major problems are from severe interference or moderate interference with low-flow velocity, which can be minimised/eliminated by the integration of the individual systems involved.

Changes to the quantity and processing of starchy foods in a western diet can increase polysaccharides escaping digestion and improve in vitro fermentation variables.

This study investigated how readily achievable changes to the quantity and processing of starchy foods in a typical Western diet: (1) were reflected in levels of resistant starch (RS) and NSP excreted from the small intestine; and (2) more favourable profiles of butyrate, NH3 and phenol production. Two diets, a low-starch diet (LSD) and a high-starch, low-fat diet (HSLFD) were compared. The LSD with 20% total energy (%E) from starch was based on a 'typical' Australian diet, while the HSLFD (40%E as starch) was the same Australian diet modified by an increased content of legumes, starchy foods and coarsely-ground cereals and by a reduced fat content. Four subjects with iliostomies consumed each diet for 2 d, with ileal effluent collection on the second day. On the HSLFD compared with the LSD, RS in ileal effluent increased from from 0.49 to 1.7 g/MJ per d (P < 0.005) while ileal NSP excretion increased from 2.0 to 3.3 g/MJ per d (P < 0.05). Ileal effluents obtained after each diet were incubated for 24 h in vitro with a human faecal innoculum. After fermentation, ileal effluent from the HSLFD produced more butyrate relative to other short-chain fatty acids (17.5 v. 15.8 molar %, P < 0.005) and less phenol (2.3 v. 5.7 mg/l, P < 0.05) and NH3 (20.3 v. 23.1 mmol/l, P < 0.005) than the LSD diet. The HSLFD also generated a lower pH (6.15 v. 6.27, P < 0.05). On a wt/wt basis, RS was 2.3-fold higher in the HSLFD effluent while NSP did not increase, suggesting that the change in RS largely contributed to the fermentation effects. Changes in in vitro variables when the HSLFD ileal effluent was ground before fermentation indicated the importance of physical structure in determining ileal excretion of RS. We conclude that: (1) readily achievable modifications to the amount and processing of starchy foods in an Australian diet would produce potential benefits for in vitro fermentation variables; and (2) the physical structure of grains and cereals is important in determining access by colonic bacteria to a carbohydrate substrate.

Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo.

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

Limited humoral immunity in hepatitis C virus infection.

BACKGROUND & AIMS: The extremely high rate of chronicity to hepatitis C virus (HVC) infection suggests an inefficient immune response. The humoral immune response to HCV was evaluated in 60 patients with chronic HCV infection and in 12 patients acutely infected with HCV. METHODS: A number of recombinant HCV antigens including the core, envelope 2 (E2), nonstructural (NS) 3, NS4, and NS5 proteins, and NS4a and E2-HVR-1 peptides were used in enzyme-linked immunoassays. RESULTS: Immunoglobulin (Ig) G antibody responses to these viral antigens, except for the HCV core, were highly restricted to the IgG1 isotype. The prevalence of antibodies of the IgG1 isotype specific for the HCV core, E2, E2-HVR1, NS3 (helicase domain), NS4, and NS5 antigens was 97%, 98%, 28%, 88%, 33%, and 68%, respectively. Antibodies of the IgG3 isotype specific for E2, E2-HVR-1, NS3, NS4, and NS5 were detected in a minority of serum samples. The IgG2 and IgG4 isotypes were rarely if ever detected. Furthermore, antibody responses to HCV viral antigens were of relatively low titer and, with the exception of anti-HCV core, were delayed in appearance until the chronic phase of infection. CONCLUSIONS: The IgG1 restriction, low titer, and delayed appearance of antibody responses elicited during HCV infection suggest that the immunogenicity of HCV proteins is limited in the context of natural infection. Inasmuch as recombinant HCV viral antigens perform as relatively normal immunogens in small animals, we suggest that the defective humoral immune responses during HCV infection may be attributable to an "immune avoidance" strategy.

Santalbic acid from quandong kernels and oil fed to rats affects kidney and liver P450.

Kernels of the plant Santalum acuminatum (quandong) are eaten as Australian 'bush foods'. They are rich in oil and contain relatively large amounts of the acetylenic fatty acid, santalbic acid (trans-11-octadecen-9-ynoic acid), whose chemical structure is unlike that of normal dietary fatty acids. When rats were fed high fat diets in which oil from quandong kernels supplied 50% of dietary energy, the proportion of santalbic acid absorbed was more than 90%. Feeding quandong oil elevated not only total hepatic cytochrome P450 but also the cytochrome P450 4A subgroup of enzymes as shown by a specific immunoblotting technique. A purified methyl santalbate preparation isolated from quandong oil was fed to rats at 9% of dietary energy for 4 days and this also elevated cytochrome P450 4A in both kidney and liver microsomes in comparison with methyl esters from canola oil. Santalbic acid appears to be metabolized differently from the usual dietary fatty acids and the consumption of oil from quandong kernels may cause perturbations in normal fatty acid biochemistry.

Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein.

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and beta-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.

Dietary intake and faecal excretion of carbohydrate by Australians: importance of achieving stool weights greater than 150 g to improve faecal markers relevant to colon cancer risk.

OBJECTIVES: This study investigated, on 53 Australians consuming a typical Western diet, the relationship between dietary intake, faecal excretion of carbohydrate and changes in faecal markers believed to be relevant to colon cancer risk, for example faecal output, transit time and concentrations of phenols, ammonia and butyrate. DESIGN: Fifty-three subjects consuming their usual diet were asked to record and weigh all food consumed for a seven day period, and to collect faeces for three days during this period. SETTING: Geelong, Victoria, Australia. SUBJECTS: All volunteers were either staff and students of the university, or associates of the authors. INTERVENTIONS: None. RESULTS: Volunteers had the following dietary intakes of carbohydrate (g/d; mean +/- s.d.); starch 131 +/- 41 (including resistant starch (RS), 5 +/- 2), sugar 108 +/- 37 and non-starch polysaccharides (NSP) 14 +/- 7. Daily faecal output was 127 +/- 70 g and transit time 47 +/- 19 h. Analysis of faecal samples found 0.8 +/- 1.2 g RS and significant relationship with the concentration (mmol/L) of butyrate excreted n faeces (r = 0.34, P < 0.05). Dietary intake of RS was associated with higher concentrations of faecal ammonia (r = 0.34, P < 0.05), but this association was reversed when RS was combined with NSP in the diet (r = 0.07, NS). In contrast to dietary intake, the faecal excretion of RS was negatively related to faecal ammonia concentration (r = -0.40, P < 0.01) and positively related to faecal output (r = 0.64, P < 0.01). Individuals who consumed more NSP in their diet (19 +/- 7 g/d) excreted more than 150 g faeces per day and had higher quantities of faecal-RS and -NSP; faster transit times; higher concentrations of short chain fatty acids and lower concentrations of potentially harmful ammonia and phenols. CONCLUSIONS: The combination of RS and NSP in the colon may be required to achieve an optimal luminal environment conducive to 'colonic health'. The results also support the suggestion that faecal output (< or > 150 g/d) may provide a useful index of colon cancer risk. High faecal outputs are achieved through higher intakes of NSP (the major component of dietary fibre).

Interferon-alpha treatment induces delayed CD4 proliferative responses to the hepatitis C virus nonstructural protein 3 regardless of the outcome of therapy.

The proliferative responses to a hepatitis C virus (HCV) recombinant nonstructural protein 3 (rNS3) were analyzed in 9 patients with chronic HCV infection before, during, and after 24 weeks of treatment with interferon-alpha (IFN-alpha) alone or in combination with ribavirin. Regardless of the therapy and the subsequent outcome, all patients showed an increased rNS3-specific proliferative response in peripheral blood mononuclear cells in vitro within 48 weeks from the start of therapy (P < .01). The proliferating cell phenotype was CD4 and was dependent on HLA-DP/DQ/DR class II antigen presentation. rNS3 induced in vitro detectable interleukin (IL)-2, IL-10, and IFN-gamma production in some patients before or after therapy (or both). No significant differences existed between responders and relapsed responders plus nonresponders with respect to the NS3-specific CD4 T helper (Th) cell responses. Thus, IFN-alpha therapy induces HCV NS3-specific CD4 Th cell proliferation regardless of the outcome of therapy.

Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus.

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.

Antibody production against hepatitis C virus core and nonstructural 3 proteins is highly sensitive to deficits in T-cell function.

The influence of suppression of CD4+ and CD8+ T cells on the humoral responses to hepatitis C virus (HCV) core and nonstructural 3 proteins was studied. An increasing viral burden cannot substitute for the lack of functional T cells in maintaining humoral HCV-specific responses.

Immunogenicity and antigenicity of the ATPase/helicase domain of the hepatitis C virus non-structural 3 protein.

The immunogenicity and antigenicity of an enzymatically functional (ATPase/helicase) recombinant protein encompassing residues 1207-1612 of the hepatitis C virus (HCV) non-structural 3 (NS3) protein was characterized using B10 congenic mice. Previous studies have indicated a high frequency of NS3-specific antibodies in HCV-infected humans. Similarly, all six immunized murine haplotypes were antibody responders to the NS3 ATPase/helicase domain, with the H-2k and H-2s haplotypes as high responders. As also observed in HCV-infected humans, the murine NS3 antibodies were predominantly directed to conformational determinants. Irrespective of the murine haplotype, IgG1 predominated in the primary anti-NS3 response, whereas IgG1 and IgG2b predominated in the secondary response. The antibody responder hierarchy was reiterated at the T cell level, with the H-2k and the H-2s haplotypes as the best responders. In both the H-2d and H-2k haplotypes ATPase/helicase-primed T cells secreted interleukin 2 and interferon gamma, corroborating observations from HCV-infected humans. In the H-2d, H-2k and H-2s haplotypes the fine specificity of the T cell recognition of the ATPase/helicase domain was further characterized. Multiple, although generally weak, T cell recognition sites were found for all three haplotypes. The large size of the NS3 protein together with the presence of multiple class II binding motifs explain the high prevalence of NS3 antibodies in immunized mice and predict a similar explanation for the observed high frequency of NS3-specific antibodies in HCV-infected humans.