David Kobiler

Search for Correlates of Protective Immunity Conferred by Anthrax Vaccine

ABSTRACT Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r2 = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD50) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Attenuated Nontoxinogenic and Nonencapsulated Recombinant Bacillus anthracis Spore Vaccines Protect against Anthrax

ABSTRACT Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 × 107 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (≥100 μg/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracisspore-associated antigen(s) may contribute in a significant manner to protective immunity.

West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice.

SummaryThe encephalitic West Nile virus and its nonneuroinvasive variant, WN-25, were used to study the effect of macrophage depletion on viral invasion of the central nervous system. The in vivo elimination of macrophages was achieved by use of liposome-encapsulated drug dichloromethylene diphosphonate. Depletion of macrophages had an exacerbating effect on the course of the viral infection, exhibited by higher and extended viremia and accelerated development of encephalitis and death. Using a low dose of West Nile virus (5 PFU/mouse), an increase in mortality (from 50% to 100%) due to macrophage depletion was demonstrated. Furthermore, the attenuated noninvasive variant WN-25 showed high and prolonged viremia in the macrophage depleted mice (≈5 log 10 PFU/ml versus 2 in control mice), that allowed the penetration of the virus into the central nervous system. The mortality rate caused by the attenuated virus in the macrophage-depleted mice was 70–75%, as compared to complete survival in the control inoculated mice. These results indicate a significant role of macrophages in the non-specific immediate defence system of the organism in case of viral infection.

Efficiency of protection of guinea pigs against infection with Bacillus anthracis spores by passive immunization.

ABSTRACT The efficacy of passive immunization as a postexposure prophylactic measure for treatment of guinea pigs intranasally infected with Bacillus anthracis spores was evaluated. Antisera directed either against the lethal toxin components (PA or LF) or against a toxinogenic strain (Sterne) were used for this evaluation. All antisera exhibited high enzyme-linked immunosorbent assay titers against the corresponding antigens, high titers of neutralization of cytotoxicity activity in an in vitro mouse macrophages cell line (J774A.1), as well as in vivo neutralization of toxicity when administered either directly to Fisher rats prior to challenge with the lethal toxin or after incubation with the lethal toxin. In these tests, anti-LF antiserum exhibited the highest neutralization efficiency, followed by anti-Sterne and anti-PA. The time dependence and antibody dose necessary for conferring postexposure protection by the various antibodies of guinea pigs infected with 25 50% lethal doses of Vollum spores was examined. Rabbit anti-PA serum was found to be the most effective. Intraperitoneal injections of anti-PA serum given 24 h postinfection protected 90% of the infected animals, whereas anti-Sterne and anti-LF were less effective. These results further emphasizes the importance of anti-PA antibodies in conferring protection against B. anthracis infection and demonstrated the ability of such antibodies to be effectively applied as an efficient postexposure treatment against anthrax disease.

Loss of active neuroinvasiveness in attenuated strains of West Nile virus : pathogenicity in immunocompetent and SCID mice

SummaryThe neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

Protective Antigen as a Correlative Marker for Anthrax in Animal Models

ABSTRACT The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.

Immunological Correlates for Protection against Intranasal Challenge of Bacillus anthracis Spores Conferred by a Protective Antigen-Based Vaccine in Rabbits

ABSTRACT Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 × 105 conferred protection, whereas lower titers (between 104 and 105) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.

Infection of Glioma Cells with Sindbis Virus Induces Selective Activation and Tyrosine Phosphorylation of Protein Kinase C δ IMPLICATIONS FOR SINDBIS VIRUS-INDUCED APOPTOSIS

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCδ to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCδ inhibitor rottlerin and a PKCδ kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCδ in the apoptosis induced by SVNI we used a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). PKCδ5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCδ. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCδ5 but did not induce cleavage of PKCδ or PKCδ5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCδ exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCδ on specific tyrosines is required for this function.

Postexposure Prophylaxis against Anthrax: Evaluation of Various Treatment Regimens in Intranasally Infected Guinea Pigs

ABSTRACT The efficiency of postexposure prophylaxis against Bacillus anthracis infection was tested in guinea pigs infected intranasally with either Vollum or strain ATCC 6605 spores (75 times the 50% lethal dose [LD50] and 87 times LD50, respectively). Starting 24 h postinfection, animals were treated three times per day for 14 days with ciprofloxacin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole (TMP-SMX). Administration of cefazolin and TMP-SMX failed to protect the animals, while ciprofloxacin, tetracycline, and erythromycin prevented death. Upon cessation of treatment all erythromycin-treated animals died; of the tetracycline-treated animals, two of eight infected with Vollum and one of nine infected with ATCC 6605 survived; and of the ciprofloxacin group injected with either 10 or 20 mg/kg of body weight, five of nine and five of five animals, respectively, survived. To test the added value of extending the treatment period, Vollum-infected (46 times the LD50) animals were treated for 30 days with ciprofloxacin or tetracycline, resulting in protection of eight of nine and nine of nine animals, respectively. Once treatment was discontinued, only four of eight and five of nine animals, respectively, survived. Following rechallenge (intramuscularly) of the survivors with 30 times the LD50 of Vollum spores, all ciprofloxacin-treated animals were protected while none of the tetracycline-treated animals survived. In an attempt to confer protective immunity lasting beyond the termination of antibiotic administration, Vollum-infected animals were immunized with a protective antigen (PA)-based vaccine concurrently with treatment with either ciprofloxacin or tetracycline. The combined treatment protected eight of eight and nine of nine animals. Following cessation of antibiotic administration seven of eight and eight of eight animals survived, of which six of seven and eight of eight resisted rechallenge. These results indicate that a combined treatment of antibiotics together with a PA-based vaccine could provide long-term protection to prevent reoccurrence of anthrax disease.

Long-term culture of infant leukemia cells: dependence upon stromal cells from the bone marrow and bilineage differentiation

Infant leukemia cells with 46XY,t(11; 17)(q23; p13) karyotype and a hybrid pre B myeloid phenotype (HLA-DR, (Ia), B4 and My7-positive and CALLA and T11-negative) and immunoglobulin heavy chain gene rearrangement were maintained in long-term culture for over 10 months. The in-vitro survival and growth of the leukemia cells were strictly dependent upon the presence of their autologous marrow stromal cells. The latter could be replaced by the 14F1.1 clone of preadipocytes derived from mouse bone marrow. Neither heterologous human marrow or foreskin fibroblasts nor fibroblast or endothelial like cell lines from mouse stroma could mimic the effect of autologous stroma or 14F1.1 adipocytes. The leukemia cells maintained their original phenotype throughout the 10-month culture period with either their autologous stroma or the 14F1.1 adipocytes. They could be induced to differentiate in two distinct directions. Phorbol myristate acetate induced adherence of the leukemia cells and development of macrophage properties. In contrast, conditioned medium from a hybridoma producing B-cell growth factor caused aggregation of the leukemia cells and expression of CALLA antigen and surface IgM. This bipotency of the leukemia cells and their dependence upon marrow stroma are properties in common with stem cells.