Lesley McLain

Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus

A plant virus, cowpea mosaic virus, expressing a 22 amino acid peptide 731-752 of the gp41 glycoprotein of human immunodeficency virus type 1 (HIV-1 IIIB), was shown previously to stimulate HIV-1 cross reactive neutralizing antibodies in adult C57/BL6 mice. Here some parameters concerning the stimulation of HIV-1-specific neutralizing and ELISA antibody have been determined in adult C57/BL6, C3H/He-mg and BALB/c mice. Two injections per mouse of all CPMV-HIV/1 doses tested (100, 10 and 1 microgram chimera which contained, respectively, 1700, 170 and 17 ng HIV peptide per injection) stimulated a strong serum neutralizing antibody response in all mice. One hundred micrograms or 10 micrograms CPMV-HIV/1 per injection gave 99% neutralization of HIV-1 IIIB in C8166 cells at a serum dilution of 1/200, whereas sera from mice immunized with 1 microgram per injection neutralized virus to 97%, 79% and 63% at a 1/200 dilution of serum from C3H/He-mg, C57/BL6 and BALB/c mice, respectively. Restimulation of these mice with the same immunogen dose marginally increased the neutralization titres. The longevity of the neutralizing antibody response increased as the immunogen dose decreased, and was dependent on the strain of mouse, in the order C57/BL6C3H/He-mg BALB/c. Re-immunization with a third injection improved the longevity of the antibody response. All mice immunized with 100 micrograms CPMV-HIV/1 responded with ELISA antibody to the gp41 peptide bound in solid phase. Ten micrograms stimulated ELISA antibody in some but not all mice, while mice immunized with 1 microgram had no detectable ELISA antibody. This synthesis of ELISA antibody decreased > or = 230-fold over the range of immunogen doses tested but, in the same mice, the neutralizing antibody response decreased only twofold, showing an unusual bias to production of the latter. Neutralizing antibodies were thus stimulated at a lower immunogen dose than ELISA antibodies. Antibody which was affinity purified using the free gp41 peptide gave a good ELISA titre but did not neutralize HIV-1, suggesting that the neutralizing antibody is recognizing a conformational epitope on the gp41 oligomer.

Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization

Control of pandemic human immunodeficiency virus type 1 (HIV-1) infection ideally requires specific mucosal immunity to protect the genital regions through which transmission more often occurs. Thus a vaccine that stimulates a disseminated mucosal and systemic protective immune response would be extremely useful. Here we have investigated the ability of a chimeric plant virus, cowpea mosaic virus (CPMV), expressing a 22 amino acid peptide (residues 731-752) of the transmembrane gp41 protein of HIV-1 IIIB (CPMV-HIV/1), to stimulate HIV-1-specific and CPMV-specific mucosal and serum antibody following intranasal or oral immunization together with the widely used mucosal adjuvant, cholera toxin. CPMV-HIV/1 has been shown previously to stimulate HIV-1-specific serum antibody in mice by parenteral immunization. All mice immunized intranasally with two doses of 10 microg of CPMV-HIV/1 produced both HIV-1-specific IgA in faeces as well as higher levels of specific, predominantly IgG2a, serum antibody. Thus there was a predominantly T helper 1 cell response. All mice also responded strongly to CPMV epitopes. Oral immunization of the chimeric cowpea mosaic virus was less effective, even at doses of 500 microg or greater, and stimulated HIV-1-specific serum antibody in only a minority of mice, and no faecal HIV-1 specific IgA.

Single- and multi-hit kinetics of immunoglobulin G neutralization of human immunodeficiency virus type 1 by monoclonal antibodies

A quantal assay, based on syncytium formation in the human T cell leukaemia-derived C8166 cell line, was used to determine the kinetics of human immuno-deficiency virus type 1 (HIV-1) strain IIIB neutralization. Three rat monoclonal antibodies (MAbs) were used, under physiological conditions of temperature and antibody concentration. MAb ICR39.3b (IgG2b) neutralized virus with no lag period while the other two MAbs, ICR39.13g (IgG2b) and ICR41.1i (IgG2a), neutralized with lag periods of 5 min and 15 min respectively. It was calculated that the latter two MAbs mediated neutralization by about two and three molecules of IgG per virion respectively. The highest neutralization rate constant (for MAb ICR 41.1i) was over 300-fold less than that of MAbs specific for the haemagglutinin of the enveloped influenza virus type A and for poliovirus type 1.

Protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication

Intranasal administration of defective interfering (DI) influenza virus (A/WSN) ensured the survival of 80% of C3H mice otherwise lethally infected with WSN by the intranasal route, whereas a control group which received beta-propiolactone-inactivated DI virus in place of DI virus died at 7.4 days post-infection. DI virus-treated mice developed significantly less lung consolidation than controls although qualitatively the cellular pathology in the two groups was indistinguishable. Surprisingly, in view of the accepted mode of action of DI virus interference, multiplication of infectious virus in the lung, production of viral haemagglutinin (HA) antigen and neuraminidase, and the distribution and amount of viral antigen in cells as shown by immune labelling were unaffected by the presence of active DI virus. Furthermore, assays of lung extracts showed that DI virus was not stimulating significantly greater amounts of interferon than the control inactivated DI virus. An alternative explanation arises from the fact that the pathology of influenza in inbred mice is immune (T lymphocyte)-mediated. Thus, since there is no evidence that DI virus affects virus multiplication we suggest that DI virus is responsible for ameliorating the damaging host responses. Another aspect of the immune response modulated by DI virus was the enhancement of local haemagglutination-inhibiting (HI) antibody in the lung, with peak increases of up to 10-fold over the relevant controls being demonstrated at 5 days after infection. This antibody was presumably complexed to HA antigen in the lung as activity was only demonstrated after elution at low pH. It had no detectable neutralizing activity (less than 10% HI: neutralization ratio of convalescent serum) which accounts for the coexistence of local antibody and virus infectivity. Mice infected with virus alone or which received beta-propiolactone-inactivated DI virus in addition to a lethal dose of WSN did not develop significant amounts of lung antibody. No differences were seen in serum HI titres. The increased level of antibody could not be attributed to the presence of greater amounts of HA antigen in lungs of mice treated with DI virus, as ELISA showed no significant difference from control preparations. The possibility that the two modulated immune responses are linked through HI antibody blocking access of T cells to cell membrane-borne HA antigen is discussed.

The neutralizing antibody response against a conserved region of human immunodeficiency virus type 1 gp41 (amino acid residues 731-752) is uniquely directed against a conformational epitope

Amino acids 731-752 (731PRGPDRPEGIEEEGGERDRDRS752) of the transmembrane glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) are conserved in most virus isolates and are controversially reported to be implicated in virus neutralization. The humoral response in infected patients against this region is poor and humans immunized with gp160 show high levels of antibodies against the peptide but poor neutralization titres. Nonetheless, several groups have succeeded in obtaining neutralizing antibodies against this sequence using different antigen-presenting systems. In order to identify the sequence(s) against which the neutralizing response was generated, we used the flock house virus (FHV) antigen-presenting system to analyse neutralizing antisera from mice immunized with a cowpea mosaic virus (CPMV) chimera expressing the 731-752 sequence. Data show that the neutralizing response is uniquely directed against a conformational epitope mapping to the ERDRD portion of this sequence, although the major antibody response, which is non-linear, and is not neutralizing, is against an IEEE epitope. These results provide an explanation for the controversy regarding the immunogenicity of this region of gp41 and suggest that this conformational epitope, in the absence of the non-neutralizing epitope, should be considered for a subunit vaccine. In addition, this study highlights the usefulness of antigen-presenting systems that preserve epitope conformation in the investigation of immune responses.

TWO NEUTRALIZING ANTI-V3 MONOCLONAL ANTIBODIES ACT BY AFFECTING DIFFERENT FUNCTIONS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

Monoclonal antibody (MAb) ICR41.1i (rat IgG2a) is specific for a conformation-dependent epitope of human immunodeficiency virus type 1 (HIV-1) V3 , and MAb F58 (mouse IgG1) recognizes the peptide IXXGPGR, at the tip of the V3 loop. Both MAbs neutralized HIV-1 strain IIIB in C8166 and HeLa-T4(CD4) cells. Neutralization by either MAb did not inhibit attachment of virus to target cells as determined by FACS analysis, ELISA or immunofluorescence, and such attachment was absolutely dependent on the availability of CD4 molecules. F58 inhibited virus-induced cell-cell fusion, and reduced internalization of virions in direct proportion to neutralization. In contrast, ICR41.li had no effect on HIV-1-mediated cell fusion or on internalization of virus. It was concluded that MAb F58 neutralized infectivity by inhibiting fusion of the virus with the cell and internalization of the viral core, and that ICR41.1i neutralized by inhibiting a post-fusion-internalization event. The possible mechanism by which a neutralizing antibody binds to the V3 loop and affects the function(s) of structures inside the virion is discussed. Lastly, postattachment neutralization (PAN) was investigated. F58 mediated PAN at 21 degrees C and 35 degrees C. However, ICR41.1i gave PAN at 21 degrees C but not at 35 degrees C, suggesting that a temperature-dependent event affecting the V3 loop had abrogated neutralization. Overall, it appears that antibodies to different epitopes within the V3 loop neutralize by affecting very different functions of the virus.

One defective interfering particle per cell prevents influenza virus-mediated cytopathology: an efficient assay system

The titre of defective interfering (DI) influenza virus measured by an assay based on the inhibition of cytopathology caused by A/WSN (H1N1) influenza virus in MDCK cells was 320,000-fold greater than titres measured by inhibition of infectious centre formation. Interference was less in other types of cell. By electron microscopy, we have shown that the ratio between physical particles and DI units in preparations of the DI virus was approximately unity, which suggested that one or few DI particles is/are required to confer resistance of a MDCK cell to viral cytopathology. This human DI virus interfered heterotypically with an avian H7N1 influenza virus.

Intracellular stability of the interfering activity of a defective interfering influenza virus in the absence of virus multiplication

Half-lives of the interfering activity of a human defective interfering (DI) influenza virus and of viral RNA in MDCK cells inoculated with noninfectious DI virus were 25 and 17 days, respectively, and of viral RNA in cells inoculated with noninfectious uv-irradiated standard virus was 21 days. In neither case was there evidence of virus replication (de novo synthesis of viral proteins, haemagglutinin, or infectivity). The half-life in BHK cells was shorter, although still considerable, showing evidence of a host contribution to stability. Implications of the putative persistence of influenza virus genes in vivo to the natural history of the virus are discussed.

Protection of mice from lethal influenza by adoptive transfer of non-neutralizing haemagglutination-inhibiting IgG obtained from the lungs of infected animals treated with defective interfering virus.

The lungs of C3H/HeMg mice infected with a lethal dose of the influenza virus A/WSN (H1N1) contained antibody to the viral neuraminidase glycoprotein but not to the haemagglutinin (HA). When coinoculated with a life-saving amount of defective interfering (DI) WSN, lungs were found to contain both anti-neuraminidase and haemagglutination-inhibiting (HI) antibodies which peaked at 3 and 5 days post-infection (p.i.), respectively. Mice coinoculated with WSN and beta-propiolactone-inactivated DI WSN had only anti-neuraminidase antibody in the lungs. The HI activity was unusual in that there was no detectable neutralizing activity. The HI activity has been affinity-purified by adsorption to and elution from WSN HA and consisted entirely of IgG, comprising isotypes IgG1, IgG2a and IgG2b. The antibody was specific for WSN HA, of low activity and had disappeared from the lungs by 20 days p.i. Transfer of affinity-purified HA-specific, non-neutralizing antibody to mice 24 h before infection with WSN alone protected 60% from death. We conclude that the antibody contributes significantly to the life-saving activity of DI WSN virus and that the ability to stimulate such antibody represents a novel property of this DI virus.

Protection of three strains of mice against lethal influenza in vivo by defective interfering virus

This report examines the protective effects of defective interfering (DI) WSN on three strains of mice (C3H/He-mg (H-2k), C57BL/6 (H-2b) and BALB/c (H-2d)) infected with various doses of A/WSN influenza virus. All three strains were protected in terms of morbidity and mortality, to varying extents, DI WSN protected optimally against a low but lethal dose of A/WSN in C3H/He-mg mice, but also protected this and other strains against very high doses of A/WSN. Intermediate sized inocula gave little, if any, protection. In all cases protection required an active DI genome since inactivation with beta-propiolactone abrogated any sparing effect. Consolidation of the lungs was reduced by treatment with active DI virus, but at some doses of inoculum there was reduction in lung pathology without reduction of mortality. Treatment of infected mice with DI virus did not reduce the lung virus titre, but in C3H/He-mg mice resulted in recovery of infectious virus from other tissues, notably the heart, where it was not normally found. No infectivity was recovered from brain, liver or serum. Haemagglutination-inhibiting (HI) antibody could not be detected in the lungs of any of the infected mice co-inoculated with the control BPL-inactivated DI WSN but was present in considerable amounts in all three strains when these were co-inoculated with DI virus. These and previous data (Morgan and Dimmock, 1992) suggested that influenza virus was immunosuppressive and that active DI virus abrogated these suppressive effects.(ABSTRACT TRUNCATED AT 250 WORDS)