Conjugation of enzymes to antibodies.

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross-linking HRPO, urease or alkaline phosphatase to immunoaffinity-purified monoclonal or polyclonal antibodies (IgG).

Mucosal and systemic antiviral antibodies in mice inoculated intravaginally with herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) causes lethal illness after intravaginal (IVAG) inoculation into BALB/cJ mice. In the present studies, we demonstrated in mice that primary IVAG vaccination with an attenuated strain of HSV-2 induced humoral immunity in sera and in vaginal secretions. Secondary genital exposure to HSV-2 enhanced this response. However, intraperitoneal exposure to attenuated HSV-2 elicited an antiviral antibody response in sera but not in vaginal secretions. In both sera and vaginal secretions, antiviral IgG antibodies were the major isotype. Systemic exposure to HSV-2 elicited antibodies only in sera that were specific for the major viral antigens whereas IVAG inoculation with HSV-2 stimulated both serum and vaginal antibody responses. Intravenous transfer of antiviral monoclonal antibodies protected against systemic HSV-2 infection but were ineffective against vaginal infection due to a lack of transudation into vaginal secretions. These results suggested that local humoral immunity in the genital tract is important in resistance to HSV-2.

Expression of immunity to intravaginal herpes simplex virus type 2 infection in the genital tract and associated lymph nodes.

Herpes simplex virus type 2 is a human venereal pathogen which causes lethal neurological illness after intravaginal inoculation into BALB/cJ mice. In the present studies, we demonstrate that intravaginal vaccination with an attenuated strain of this virus, which possesses a partial deletion of the thymidine kinase gene, rapidly induced durable immunity to lethal intravaginal challenge with wild-type virus. Such immunity was characterized by a dramatic hyperplasia of genital lymph nodes and a significant reduction in wild-type virus replication and spread from the genital tract following lethal challenge. Of greater importance, immunity to lethal wild-type virus challenge in the genital tract was transferrable to non-immune mice with genital lymph node cells prepared 1 week after intravaginal vaccination but was not transferrable with serum or cells from other lymphoid organs tested at this time. The adoptive transfer of anti-viral immunity to wild-type challenge was also characterized by a diminution in wild-type virus replication and spread from the genital tract. These results suggest that an important component of cellular immunity to genital pathogens may be antigenic stimulation of genital lymph nodes.

Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase.

The herpes simplex virus ribonucleotide reductase is associated with two viral proteins which are both immunoprecipitated by monoclonal antibodies specific for the enzyme. We separated the two proteins and showed that individual antibodies react solely with one or the other. In addition, antibodies to either protein can neutralize enzymatic activity. Our data demonstrate that the proteins are associated in a complex and constitute the subunits of the enzyme.

Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity.

Mammalian cells infected with herpes simplex virus (HSV) express a novel ribonucleotide reductase which is biochemically and immunologically distinct from the uninfected-cell enzyme. Using polyvalent rabbit antiserum raised against partially purified HSV type 2 reductase as well as monoclonal antibodies to HSV type 1 and HSV type 2 early antigens, we have been able to show that in both serotypes reductase activity is associated with phosphoproteins of molecular weights 144,000 and 38,000 encoded between map units 0.566 and 0.602 in the viral genomes. The major antigenic species (144,000) have been tentatively identified as HSV type 1 ICP6 and HSV type 2 ICP10.

Carrageenan stimulates the release of dialyzable helper factors.

We found that 1 mg of carrageenan (CGN) could enhance the primary antibody response of BALB/c spleen cells to sheep erythrocytes (SRBC). This enhancement was dependent on the strain of mouse injected as well as the type of CGN administered. In vitro experiments with spleen cells from mice treated with CGN in vivo showed that these cells release low molecular weight factor(s) which enhanced the in vitro antibody response to SRBC. Thy 1+ cells were clearly involved in the production of this factor since depletion of these cells eliminated the helper effect of CGN-treated spleen cells. Our results suggest one possible mechanism by which CGN can modulate the immune response.