According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.
It has been difficult to obtain pure Pneumocystis carinii antigen either from cultures or from infected lungs for use in producing a specific antibody against P. carinii. This report describes an approach toward producing a monoclonal antibody that bypasses the antigen purification steps. P. carinii infection was developed in Sprague-Dawley rats by the method of immunosuppression with cortisone. The infected lungs were homogenized, and the homogenate was used to immunize Sprague-Dawley rats. Rat spleen cells were then fused with SP2/0 mouse myeloma cells. Hybridoma clones were screened for antibody production against P. carinii by immunoperoxidase staining techniques and by enzyme-linked immunosorbent assay, 'using as antigens homogenates of normal rat lung, homogenates of P. carinii-infected rat lung, and harvests of P. carinii grown with WI-38 cells. Out of six hybridoma clones obtained that produced antibodies against P. carinii, one was able to produce ascitic fluid. This monoclonal antibody reacted with two P. carinii antigens with masses of about 35,000 and 65,000 daltons in P. carinii-infected lungs and three proteins with masses of about 35,000, 65,000, and 110,000 daltons in P. carinii that was harvested from a WI-38 cell culture. Pneumocystis carinii, a common cause of subclinical infections in healthy people, also causes severe infections in * Corresponding author. MATERIALS AND METHODS Animals and chemicals. Sprague-Dawley rats and BALB/c mice were obtained from Harlan Laboratory, Indianapolis, Ind. WI-38 cells were purchased from plow Laboratories, Inc., McLean, Va. Cortisone aipetate was obtained from Merck Sharp & Dohme, Wqst Point, Pa. Staphylococcus aureus cells, protein A, bovine serum albumin (BSA), and other common chemicals were provided by Sigma Chemical Co., St. Louis, Mo. Rabbit anti-rat immunoglobulin G (IgG) and peroxidase-conjugated rabbit anti-rat IgG were purchased from Jackson ImmunoResearch Laboratories, Inc., Avondale, Pa. Na251I was obtained from Amersharn Corp., Arlington Heights, Ill. lodo-beads were supplied by Pierce Chemical Co., Rockford, Ill.
The efficacy of a herpes simplex virus type 1 (HSV-1) envelope antigen (EAG) preparation against HSV infection was studied in T cell competent and T cell deficient mice. Immuno-competent mice were successfully protected against herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) infection when immunized 2 weeks prior to this infection with a heat-inactivated whole virus preparation or a HSV-1 envelope antigen (HSV-1 EAG) preparation. Since HSV-1 EAG was considerably less effective than the whole virus preparation, a poly.riboinosinic-poly.ribocytidylic acid complex with poly-L-lysine and carboxymethylcellulose (PICLC) was used as adjuvant. Immunization with HSV-1 EAG plus PICLC resulted in a pronounced increase of this protection rate as compared with that obtained after immunization solely with HSV-1 EAG. PICLC alone, however, offered no protection when given 2 weeks before challenge. In T cell deficient nu/nu mice no protection was achieved with HSV-1 EAG while their T cell competent, heterozygous littermates were protected. From these results it may be concluded that T cell competence is a prerequisite for establishing a protective immunity against HSV infection after immunization with HSV-1 EAG.
Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu53-Ala312) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.
Because the reported frequency of teichoic acid precipitins in controls and various patient groups has varied considerably among laboratories, we studied the effect of using various concentrations of staphylococcal extracts in agar-gel diffusion tests for teichoic acid antibodies. Of 25 normal sera, only 1 was positive against an undiluted extract, but 4 were positive against a 16-fold-diluted extract. Of nine sera from patients with staphylococcal bacteremia, two were positive at a higher titer against the diluted extract. A false-positive serum against the undiluted extract had a twofold titer increase against the diluted extract. Because human immune serum globulin is generally used as a positive teichoic acid antibody control, the variability of five different lots was studied. Three lots ahd teichoic acid antibody titers of 1:4, whereas one each had titers of 1:2 and 1:8. Based on this study, we feel that staphylococcal extracts should not be diluted. If immune serum globulins are used to determine the adequacy of ultrasonic extracts, newly acquired globulin lots should be standarized against an ultrasonic extract of proven sensitivity and specificity.
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