Sulfonate-isosteric replacement examined within heroin-hapten vaccine design.

Heroin overdose and addiction remain significant health and economic burdens in the world today costing billions of dollars annually. Moreover, only limited pharmacotherapeutic options are available for treatment of heroin addiction. In our efforts to combat the public health threat posed by heroin addiction, we have developed vaccines against heroin. To expand upon our existing heroin-vaccine arsenal, we synthesized new aryl and alkyl sulfonate ester haptens; namely aryl-mono-sulfonate (HMsAc) and Aryl/alkyl-di-sulfonate (H(Ds)2) as carboxyl-isosteres of heroin then compared them to our model heroin-hapten (HAc) through vaccination studies. Heroin haptens were conjugated to the carrier protein CRM197 and the resulting CRM-immunoconjugates were used to vaccinate Swiss Webster mice following an established immunization protocol. Binding studies revealed that the highest affinity anti-heroin antibodies were generated by the HMsAc vaccine followed by the HAc and H(Ds)2 vaccines, respectively (HMsAc > HAc≫HDs2). However, neither the HMsAc nor H(Ds)2 vaccines were able to generate high affinity antibodies to the psychoactive metabolite 6-acetyl morphine (6-AM), in comparison to the HAc vaccine. Blood brain bio-distribution studies supported these binding results with vaccine efficiency following the trend HAc > HMsAc â‰« H(Ds)2 The work described herein provides insight into the use of hapten-isosteric replacement in vaccine drug design.

The Cryptosporidium parvum gp60 glycoprotein expressed in the ciliate Tetrahymena thermophila is immunoreactive with sera of calves infected with Cryptosporidium oocysts.

Cryptosporidium parvum is a protozoan parasite of the phylum Apicomplexa responsible for cryptosporidiosis in calves, a disease that causes significant diarrhea and impairs gain of body weight, generating important production losses. As to now, no effective drugs or vaccines are available for the treatment or prevention of bovine cryptosporidiosis. Several reports suggest that development of a vaccine to prevent cryptosporidiosis is feasible, but relatively few vaccine candidates have been characterized and tested. The most prominent C. parvum antigen is gp60, an O-glycosylated mucin-like protein tethered to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor. Gp60 has been shown to be involved in essential mechanisms for the survival of C. parvum, such as recognition, adhesion to, and invasion of host cells. This work was aimed at expressing gp60 in Tetrahymena thermophila, a ciliated protozoon with numerous advantages for the heterologous expression of eukaryotic proteins, as a first approach for the development of a recombinant vaccine for bovine cryptosporidiosis. T. thermophila-expressed gp60 localized to the protozoon cell surface and oral apparatus, and partitioned into the Triton X-114 detergent phase. This indicates that the protein entered the reticuloendothelial system of the ciliate, and suggests it contains a GPI-anchor. Homogenates of gp60-expressing T. thermophila cells were recognized by sera from calves naturally infected with C. parvum demonstrating their immunoreactivity. In summary, the heterologous expression of gp60, a C. parvum-encoded GPI-anchored protein, has been successfully demonstrated in the ciliate T. thermophila.

Detection of classical swine fever vaccine virus in blood and tissue samples of pigs vaccinated either with a conventional C-strain vaccine or a modified live marker vaccine.

Attenuated live classical swine fever (CSF) viruses are the most efficacious vaccines against the disease. However, little is known about the distribution and detection of CSF vaccine viruses in the host. We therefore compared the new recombinant attenuated marker vaccine virus CP7_E2alf with the conventional C-strain vaccine concerning virus isolation, antigen-, and genome-detection in different samples within the first 42 days post-vaccination (p.v.). Leukocytes and several organs such as tonsils, lymph nodes, spleen, thymus, parotis and kidney were also tested using highly sensitive real-time reverse transcription-polymerase chain reaction (RT-PCR) techniques. It was demonstrated that vaccine virus could be detected by live animal sampling only in a few leukocytes samples at very low titres and genome copy numbers within the first 14 days after immunisation. Vaccine virus could also be isolated from individual tonsil samples within the first 6 days after vaccine application. In contrast, vaccine virus genomes were consistently detected in the tonsils up to day 42 by real-time RT-PCR. Distribution, amount of virus and viral genome levels were similar for both tested vaccines. In conclusion, blood samples could be the sample material of choice for detecting CSF wild type virus infection even in vaccinated animals after more than 14 days p.v., while tonsil sampling provided appropriate material for long-term detection of both tested CSF vaccine viruses using real-time RT-PCR methods.

Antibody response to Engerix-B and Recombivax-HB hepatitis B vaccination in end-stage renal disease.

Recombivax-HB (REC) and Engerix-B (ENG) are FDA-approved vaccines for hepatitis B virus (HBV) in end-stage renal disease (ESRD). This study compares antibody response rates between them in routine clinical practice. Patients completing the recommended 40 mug dose of REC (3 doses) or ENG (4 doses) between January 1, 2000 to April 30, 2003 were eligible. Patients with prior positive HBV surface antigen (HBsAg) or antibody (HBsAb) test results were excluded. The conversion rate and persistence of protective titer (HBsAb titer>or=10 IU/mL) were tracked for 1 year. A supplemental analysis of a one-to-one matched patient sample was also performed. REC patients (N=885) were older, had longer dialysis vintage, and had a larger proportion of whites than ENG patients (N=13,661). Cumulative conversion response was greater in ENG (58%) than REC (40%) at 1 year (p<0.0001). The odds ratio for response to ENG compared with REC was 1.96 (95% limits: 1.56, 2.45; p<0.0001) adjusted for age, gender, race, diabetes, vintage, BSA, hemoglobin, and eKt/V. Persistent protective HBsAb after 1 year was 77% (ENG) vs. 53% (REC). HBsAg was positive in 208 ENG patients (1.5%) with all but 1 because of transient, vaccine-related antigenemia. The difference in conversion response favoring ENG persisted in a one-to-one sample matched for age, gender, race, modality, and dialysis vintage. The study found higher seroconversion response to ENG compared with REC at several time points up to 1 year. Protective HBsAb disappeared in 23-47% of patients 1 year later, validating CDC recommendations to re-test HBsAb yearly. The observed difference in response rates may be related to the extra ENG dose given at the second month (0, 1, 2, 6 regimen). The study raises a hypothesis that requires confirmation in a prospective clinical trial.

Protocol for recombinant RBD-based SARS vaccines: protein preparation, animal vaccination and neutralization detection.

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

Lessons from a multisite international trial in the Caribbean and South America of an HIV-1 Canarypox vaccine (ALVAC-HIV vCP1452) with or without boosting with MN rgp120.

BACKGROUND: The first multicenter, international National Institutes of Allergy and Infectious Diseases (NIAID)-sponsored HIV vaccine trial took place in Brazil, Haiti, Peru and Trinidad. This randomized, double-blind, placebo-controlled, phase 2 trial evaluated the safety and immunogenicity of a clade B-derived, live canarypox HIV vaccine, vCP1452. vCP1452 was administered alone or with a heterologous boost of MN rgp120 glycoprotein. The trial was pivotal in deciding whether these vaccines advanced to phase 3 efficacy trials. METHODS: Forty seronegative volunteers per site were randomized to ALVAC alone, ALVAC plus MN rgp120, or placebo in a 0, 1, 3, and 6 month schedule. Immunogenicity was assayed by chromium-release cytotoxic T lymphocyte (CTL) responses; interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot assays (ELISpot); lymphocyte proliferation assays (LPA); neutralization; and enzyme-linked immunosorbent assays (ELISA). RESULTS: Enrollment and follow-up were excellent. Both vaccines were well tolerated. Neutralizing antibody to the laboratory-adapted MN strain was detected. Cellular immune responses, as measured by CTL, ELISpot, and LPA, did not differ between vaccines and placebos. CONCLUSIONS: The observation of disappointing immunogenicity in this and a parallel domestic study has informed future vaccine development. Equally important, challenges to doing an integrated trial across countries, cultures, languages, and differing at-risk populations were overcome. The identification of specific safety, ethical, logistic, and immunological issues in this trial established the foundation for current larger international studies.