Detection of HCV salivary antibodies by a simple and rapid test.

Hepatitis C (HCV) is common in developing countries, where blood sampling and expensive sophisticated methods for detection are less available. Hemodialysis patients have high prevalence of HCV and may resemble sick populations in developing countries in relation to immunosuppression and antibodies production. For these reasons anti-HCV antibodies were assayed in saliva of hemodialysis patients by ImmunoComb II assay that is less laborious, relatively inexpensive and easy to perform If the findings are confirmed by larger studies this method may be useful especially in developing countries. Serum and saliva samples were obtained from 37 hemodialysis patients and assayed by ImmunoComb II kit. In positive PCR patients the saliva test had 100% sensitivity, which was as good as serum anti-HCV Axsym testing. Saliva testing had a similar or better specificity than the serum method.

Are coping resources related to humoral reaction induced by academic stress? An analysis of specific salivary antibodies to Epstein-Barr virus and cytomegalovirus.

The aim of the present study was to investigate whether coping resources mediated the changes in Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) specific salivary antibodies caused by academic stress. Fifty-four first-year female students of nursing and physiotherapy completed pencil and paper written questionnaires and concurrently donated saliva samples. The instrument included the short version of the Sense of Coherence (SOC) scale, measures of social support, current health, health practices, the scale of psychological distress, and state anxiety questionnaire. Data and saliva samples were collected one month after the beginning of the first semester, during term examinations period and a month into the second semester. Statistically significant changes in the level of specific salivary EBV and HCMV antibodies were observed between the four study points. State anxiety and psychological distress were significantly associated with HCMV-specific salivary antibody level increase during examinations and its decrease after the stress was over. Coping resources, however, were not associated with changes in any of the antibodies studied.

Human cytomegalovirus salivary antibodies as related to stress.

Human cytomegalovirus (HCMV) is prevalent in 50-80% of the population worldwide. After primary infection it remains in a latent state until reactivation. Stressful events induce the release of corticosteroids which activate HCMV. The effect of examination stress on HCMV reactivation among first year female students was studied by detecting the values of HCMV specific salivary IgG and IgA antibodies before, during and after two important examinations. Hepatitis A virus (HAV) salivary antibodies served as a non-latent virus control. A statistically significant increase in the level of HCMV specific IgG and IgA antibodies was detected in saliva samples collected during the two examinations, as compared with the samples collected one month before them and two weeks after the grades were posted (p<0.05), whereas HAV antibody levels did not change significantly.

Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188.

Electroporation has been reported to facilitate naked DNA gene transfer in skeletal muscle, but has also been implicated in the pathogenesis of electrical injuries. To assess the effects of electroporation on gene transfer, mouse quadriceps muscles were injected with the luciferase reporter plasmid VR1255 and electroporated with caliper electrodes. Intramuscular luciferase expression was increased 10- to 70-fold by electroporation, depending on the DNA dose and injection volume used. In the absence of plasmid DNA injection, electroporation of quadriceps muscles resulted in rapid elevations in serum creatine phosphokinase activity, but did not elicit visible muscle damage. However, in muscles injected with plasmid DNA and electroporated, visible lesions consistently developed in the areas proximal to electrode placement when field strengths optimal for gene transfer (300 volts/cm) were applied. The development of muscle lesions was independent of plasmid transgene expression and required the presence of plasmid in the muscle during electroporation. Co-injection of poloxamer 188 (pluronic F68) with VR1255 substantially reduced elevations in serum creatine phosphokinase activity following electroporation, but did not inhibit the development of muscle lesions. In non-electroporated muscles, co-injection of poloxamer 188 increased luciferase expression threefold. Poloxamer 188 may thus constitute a useful excipient for intramuscular delivery of naked DNA.

Salivary HCV-antibodies; a follow-up cohort study of liver disease patients.

We have recently shown in Liver Clinic patients that saliva instead of serum may be used for anti-HCV detection. As compared to blood withdrawing, saliva is easier to obtain, non invasive, especially for infants. In the present study, sequential determination of serum and salivary anti-HCV was performed in the same cohort for 36 months. Anti-HCV seropositive and seronegative patients were studied. Blood and saliva samples were obtained simultaneously. From the anti-HCV seronegative patients (n=33), 161 sequential serum and 161 matched saliva samples were obtained. All were anti-HCV negative. From the anti-HCV seropositive patients (n=35), 131 sequential serum and 131 matched saliva samples were obtained. All sequential serum samples were anti-HCV positive. Of the saliva samples 126 (96%) were anti-HCV positive and five (4%) were anti-HCV negative. These five samples were obtained from two patients with autoimmune hepatitis and HCV-RNA seronegative by PCR. The results suggest that saliva may serve as a substitute for serum for the detection of anti-HCV antibodies.

Rabbit EPO gene and cDNA: expression of rabbit EPO after intramuscular injection of pDNA.

Erythropoietin (EPO) cDNA was cloned from kidney total RNA of a NZW rabbit. The cDNA comprises a 588-bp open reading frame encoding a 195 amino acid protein with distinguishable regions of high of homology to other mammalian EPOs. Intramuscular injection of mice with a rabbit EPO expression plasmid resulted in a significant hematocrit increase. A rabbit genomic DNA fragment was also cloned using the rabbit EPO cDNA. This 4312-bp genomic DNA fragment contains sequences homologous to the mouse EPO promoter and hypoxia-responsive enhancer. In addition, the genomic DNA also presents a high degree of conservation to other regions involved in hypoxia response. Sequence divergence in the 3' UTR may indicate differences in regulation of mRNA stability or response to low oxygen tension.

Epstein-Barr virus specific salivary antibodies as related to stress caused by examinations.

Epstein-Barr virus (EBV) is prevalent in 90% of the population. After primary infection it remains in a latent state and the majority of the virus carriers are asymptomatic during their life. Among the immunocompromized patients such as organ and bone marrow transplant recipients, individuals lacking T cell immunity, and patients treated with corticosteroid, cancer, and AIDS patients EBV primary infection and reactivation can cause life threatening diseases. Immunosupression may occur also during stressful events, which induce corticosteroid release and thus activate EBV. The effect of examination stress on EBV reactivation among female students was studied by detecting the values of EBV specific IgG and IgA salivary antibodies. Sequential saliva samples were obtained from first year female students before, during, and after two important examinations. EBV specific IgG and IgA salivary antibodies were tested by enzyme-linked immunosorbent assay (ELISA). Hepatitis A virus (HAV) salivary antibodies served as a non-latent virus control. A statistically significant increase in the values of EBV specific IgG and IgA antibodies was detected in samples collected during the examinations, as compared to the samples collected two months before and one month after the exams (P < 0.05). HAV antibody levels did not change significantly between the four time points. The menstrual cycle had no significant effect on the results. No significant symptoms were reported during the whole study. These results indicate that among female students who endure stress during academic examinations, a significant increase in EBV specific IgG and IgA salivary antibody values could be detected. EBV reactivation should be confirmed by measuring salivary EBV DNA or infectious virus.

HIV-1 IgA specific serum antibodies and disease progression during HIV-1 infection.

OBJECTIVE: To evaluate the role of serum human immunodeficiency virus type 1 immunoglobulin A (HIV-1 IgA) antibodies in the progression of HIV-1 infection in relation to viral load and CD4 cell counts. METHODS: Sequential serum specimens were obtained from 218 homosexual men: 123 HIV-1 seropositives, 24 HIV-1 seroconverters, and 71 HIV-1 seronegatives. HIV-1 IgA antibodies were tested blindly by enzyme-linked immunosorbent assay and Western blot. T-lymphocyte subsets were measured by flow cytometry. Viral plasma load was determined by a sensitive branched DNA assay. RESULTS: HIV-1 IgA antibodies with a titer greater than or equal to 50 were detected among 50% of the seroconverters, 27% of the HIV-1-seropositive asymptomatic subjects, 25% of lymphadenopathy, and 23% of HIV-1-related symptomatic subjects. Among patients with the acquired immune deficiency syndrome, the prevalence of virus-specific IgA antibodies (55%) was significantly higher (p < 0.03) as compared with the HIV-1-seropositive asymptomatic subjects, lymphadenopathy and HIV-1-related symptomatic patients, but not versus the seroconverters (p = 0.8). IgA antibodies to HIV-1 gP160 were the most prevalent among all subjects tested. A significant decrease in CD4 cell counts was observed after HIV-1 seroconversion. Viral load was slightly higher among the seroconverters who demonstrated higher (> or =50) HIV-1 IgA levels. CONCLUSIONS: HIV-1 IgA serum antibodies did not predict the progression of the disease. Correlation between HIV-1 IgA antibodies titer, viral load, and CD4 cell counts was not detected.

Comparison of seroprevalences of human herpesvirus-6 and -7 in healthy blood donors from nine countries.

BACKGROUND AND OBJECTIVES: The purpose of the study was to register antibody prevalences of HHV-7 in various locations of the world in comparison to the closely related HHV-6. MATERIALS AND METHODS: Sera of healthy blood donors from nine countries in five continents were titered by indirect immunofluorescent assays using HHV-6 infected HSB2 and HHV-7 infected SupT1 cells. RESULTS: Antibody prevalence for HHV-7 is high (75-98%) in practically all countries except for Northern Japan (44%), with no simple correlation to elevated HHV-6 antibody titers. There were regions of low, intermediate and high mean antibody titers against HHV-7 such as 78.5-91.3 for Belgium, Israel, Japan, USA and Australia, 175.4-182.6 for Mexico and Cologne/Germany, and 389.2 for South Africa for which geographic characteristics may be responsible. CONCLUSION: HHV-7, similar to HHV-6, is a widespread human herpesvirus with elevated antibody titers in the healthy human population essentially everywhere. The data warrant further studies to evaluate its possible pathologic potential, preferentially in persons with defective immune responses.

In vitro expression and in vivo immunogenicity of Plasmodium falciparum pre-erythrocytic stage DNA vaccines.

DNA vaccine plasmids were constructed that encoded four pre-erythrocytic antigens from the human malaria parasite Plasmodium falciparum: circumsporozoite protein (PfCSP); sporozoite surface protein 2 (PfSSP2); carboxyl terminus of liver stage antigen 1 (PfLSA-1 c-term); and, exported protein 1 (PfExp-1). Antigen expression was evaluated in vitro by immunoblot analysis of tissue culture cells following transient transfection with each plasmid. Clearly detectable levels of expression depended upon, or were markedly enhanced by, fusion of the antigen encoding sequences in-frame with the initiation complex and peptide leader sequence of human tissue plasminogen activator protein. Mice injected with these plasmids produced antigen specific antibody and cytotoxic T lymphocyte responses. However, the magnitudes of the responses were not always predicted by the in vitro expression assay. The results of this study provided the basis for further testing of these plasmids in primates and the formulation of multi-component pre-erythrocytic DNA vaccines for efficacy testing in human volunteers.

Hepatitis C virus infection and genotypes in Southern Israel and the Gaza Strip.

The Gaza Strip borders the southern part of Israel and Egypt. There is a remarkable difference in the prevalence of antibodies to hepatitis C virus (HCV) between Israel (0.5%) and Egypt (10%). A few thousand inhabitants cross the borders daily from the Gaza Strip to both countries. The objectives of this study were to investigate the prevalence of HCV infection in the Gaza Strip, an area that was not studied before, and to study HCV transmission in the Gaza Strip by characterizing the genotypes of HCV in Southern Israel and the Gaza Strip and comparing them with those found in Egypt. HCV prevalence in the Gaza Strip was found to be 2.2%, relatively higher than in Israel but lower than in Egypt. The most common genotypes found were type 1 b in Southern Israel and type 4 in the Gaza Strip, corresponding to the most prevalent genotype in Egypt. Similarity between type 4 isolates from the Gaza Strip and Egypt was illustrated further by sequence analysis of the HCV 5' noncoding region (NCR).

Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for protection against intracellular pathogens but often have been difficult to induce by subunit vaccines in animals. DNA vaccines elicit protective CD8+ T cell responses. Malaria-naïve volunteers who were vaccinated with plasmid DNA encoding a malaria protein developed antigen-specific, genetically restricted, CD8+ T cell-dependent CTLs. Responses were directed against all 10 peptides tested and were restricted by six human lymphocyte antigen (HLA) class I alleles. This first demonstration in healthy naïve humans of the induction of CD8+ CTLs by DNA vaccines, including CTLs that were restricted by multiple HLA alleles in the same individual, provides a foundation for further human testing of this potentially revolutionary vaccine technology.

HCV antibodies in saliva and urine.

Infection with hepatitis C virus (HCV) is usually established by detection of serum antibodies (anti-HCV). This study was conducted in order to evaluate whether saliva and urine may substitute serum for anti-HCV detection. Serum, saliva, and urine were obtained simultaneously from 141 patients with a variety of liver diseases and from 52 patients with autoimmune diseases (systemic lupus erythematosus n = 27 and rheumatoid arthritis n = 25). The cell free fraction of saliva and urine samples was tested for anti-HCV using a modification of a serum anti-HCV kit. Western blot analysis was used as a confirmation method. Of the patients with liver diseases, 73 were anti-HCV-seropositive. Salivary and urinary anti-HCV could be detected in 66 (90%) and 36 (49%) of the anti-HCV-seropositive patients, respectively. The presence of anti-HCV in saliva or urine was not related to the severity of liver disease. All the anti-HCV-seronegative liver patients were negative for salivary anti-HCV and 22 (32%) had urinary anti-HCV. The patients with autoimmune diseases were all anti-HCV-seronegative. None had detectable salivary anti-HCV while 33 (63%) were positive for urinary anti-HCV. Western Blot analysis confirmed the presence of anti-HCV in all serum and saliva samples tested but only in 2/12 urine samples. The results suggest that saliva, but not urine, may serve as a substitute for serum for the determination of anti-HCV positivity.

Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use.

Data generated in the Plasmodium yoelii rodent model indicated that plasmid DNA vaccines encoding the P.yoelii circumsporozoite protein (PyCSP) or 17 kDa hepatocyte erythrocyte protein (PyHEP17) were potent inducers of protective CD8+ T cell responses directed against infected hepatocytes. Immunization with a mixture of these plasmids circumvented the genetic restriction of protective immunity and induced additive protection. A third DNA vaccine encoding the P. yoelii sporozoite surface protein 2 (PySSP2) also induced protection. The P. falciparum genes encoding the homologues of these three protective P. yoelii antigens as well as another P. falciparum gene encoding a protein that is expressed in infected hepatocytes have been chosen for the development of a human vaccine. The optimal plasmid constructs for human use will be selected on the basis of immunogenicity data generated in mice and nonhuman primates. We anticipate that optimization of multi-gene P. falciparum DNA vaccines designed to protect against malaria by inducing CD8+ T cells that target infected hepatocytes will require extensive clinical trials during the coming years.

Malaria DNA vaccines in Aotus monkeys.

In preparation for the development of DNA vaccines designed to produce protective antibodies against Plasmodium falciparum antigens (Ag), we conducted studies to optimize antibody responses in Aotus monkeys after immunization with the P. yoelli circumsporozoite (CSP) DNA vaccine. We demonstrate in Aotus monkeys that an intradermal route of immunization with a PyCSP plasmid DNA vaccine generates antibody responses equivalent to a multiple antigen peptide/adjuvant based vaccine, and that these data support the use of the intradermal route for initial studies of the efficacy of DNA vaccines in inducing protective antibodies against P. falciparum antigens in Aotus monkeys.

Hepatitis B and C viruses serology in patients with SLE.

Sera of 95 patients with SLE were tested for the presence of hepatitis B surface antigen (HBsAg) and anti-hepatitis C antibodies. The results show that HBsAg was not detected in the sera of all of the SLE patients. Only one patient was confirmed to have anti-HCV antibodies, suggesting that chronic infection with hepatitis B and C is not increased in patients with SLE compared with the general population.

Prevalence of specific IgA and IgM anti-HBc antibodies compared with HBV DNA in the sera of HBsAg chronic carriers.

OBJECTIVE: We evaluated the significance of IgA antibodies directed against the hepatitis B virus core antigen (IgA anti-HBc) as a marker for viral replication. STUDY DESIGN/METHODS: Serum samples of 143 hepatitis B surface antigen (HBsAg) carriers and 189 HBsAg-negative subjects were studied. Hepatitis B virus (HBV) DNA was detected by polymerase chain reaction. IgA anti-HBc was determined by a capture enzyme-linked immunosorbent assay developed in our laboratory. The results were compared with those for IgM anti-HBc, which were determined by a commercially available method. RESULTS: IgA anti-HBc was detected in 57 (40%) and HBV DNA in 38 (27%) of the HBsAg carriers. Among the HBsAg-negative subjects, IgA anti-HBc and HBV DNA were detected simultaneously in four samples. All 42 HBV DNA-positive samples were IgA anti-HBc positive. IgM anti-HBc was detected in 27 (64%) of them. CONCLUSIONS: IgA anti-HBc is a sensitive marker for HBV replication, and its absence may exclude HBV replication. The role of IgA anti-HBc in monitoring response to therapy and predicting clinical course is being evaluated.

DNA vaccines against malaria: immunogenicity and protection in a rodent model.

Since the first demonstration of the technology a few years ago, DNA vaccines have emerged as a promising method of vaccination. In a variety of experimental systems, DNA vaccines have been shown not only to induce potent immune responses, but also to offer many advantages in terms of ease of construction, testing, and production. In this article we summarize the progress achieved in development of DNA vaccines that can protect mice from infection by the rodent malaria parasite Plasmodium yoelii, describe initial studies of immunogenicity of a malaria DNA vaccine in a primate model, and outline the strategies being employed to design the next generation of malaria DNA vaccines.