Karl Eduard Schneweis

Anti-glycoprotein B monoclonal antibody protects T cell-depleted mice against herpes simplex virus infection by inhibition of virus replication at the inoculated mucous membranes.

A monoclonal antibody (MAb 2c) specific for glycoprotein B of herpes simplex virus (HSV) mediated clearance of the virus from the infected mucous membranes. Young adult C57BL/6J mice were inoculated intravaginally with HSV type 1 and injected intraperitoneally either 24 and 72 h or 65 and 265 h post-inoculation with a polyclonal immune serum or the MAb 2c, both adjusted to the same neutralizing capacity. Immunization with the polyclonal immune serum did not alter the duration of virus shedding from the genital mucous membranes although a lethal outcome of infection was clearly prevented. Immunization with the MAb, however, resulted in a rapid clearance of the virus from the genital tract thus completely inhibiting genital inflammation and lethality. The same effects were achieved in mice depleted in vivo of CD4+ T cells although peripheral virus replication continued longer in these mice. In mice depleted of both CD4+ and CD8+ T cells the polyclonal immune serum was no longer able to protect against lethality, and virus replication in the mucous membranes persisted until the mice died. In contrast, after treatment with the MAb peripheral infection was quickly eliminated and all mice survived. These findings indicate that clearance of the virus from the primary site of replication can be mediated by humoral immunity. The relevance of this observation for vaccination against HSV is discussed.

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody

Despite the availability of antiviral chemotherapy, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections remain a severe global health problem. Of particular concern is the growing incidence of drug resistance in immunocompromised patients, which stresses the urgency to develop new effective treatment alternatives. We have developed a humanized monoclonal antibody (mAb hu2c) that completely abrogates viral cell-to-cell spread, a key mechanism by which HSV-1/2 escapes humoral immune surveillance. Moreover, mAb hu2c neutralized HSV fully independent of complement and/or immune effector cell recruitment in a highly efficient manner. Prophylactic and therapeutic administration of mAb hu2c completely prevented infection-related mortality of severely immunodeficient mice being challenged with a lethal dose of HSV-1. The high neutralization capacity of mAb hu2c was fully maintained toward clinical HSV isolates being multiresistant to standard antiviral drugs, and infection was fully resolved in 7/8 nonobese diabetic/SCID mice being infected with a multidrug resistant HSV-1 patient isolate. Immunohistochemical studies revealed no significant cross-reactivity of the antibody toward human tissues. These features warrant further clinical development of mAb hu2c as an immunotherapeutic compound for the management of severe and particularly drug-resistant HSV infections.

Protection against parenteral HIV-1 infection by homozygous deletion in the C-C chemokine receptor 5 gene

OBJECTIVES To investigate the role of the CC chemokine receptor 5 (CCR5) for parenteral transmission of HIV-1. DESIGN The prevalence of the delta32 deletion within the CCR5 gene was determined in a cohort of 207 patients, who had received documented amounts of non-antibody-tested and non-inactivated clotting factor concentrate. METHODS Chromosomal DNA of haemophiliacs was isolated from whole blood. A portion of the CCR5 gene spanning the delta32 deletion was amplified by PCR. The resulting DNA fragments were analysed by agarose gel electrophoresis. RESULTS The rate of HIV-1 infection was correlated strongly with increasing amounts of inoculated clotting factor concentrate. None of the HIV-positive patients (n = 129) had the delta32/delta32 genotype, whereas 12 out of 78 HIV-negative haemophiliacs had the homozygous delta32 deletion. CONCLUSIONS The delta32/delta32 genotype was highly protective against HIV-1 infection, even in patients who had received millions of non-inactivated clotting factor units. As it is likely that in the early 1980s plasma pools were contaminated not only with monocyte-tropic HIV-1 strains, CCR5 appears to be the major mediator of HIV-1 infection. Furthermore, we conclude that there must be other protective mechanisms in multiply exposed non-infected haemophiliacs who have wild-type CCR5.

Graded cytopathogenicity of the human immunodeficiency virus (HIV) in the course of HIV infection

To determine whether the biological variability of HIV-1 has any clinical significance, the highly variable cytopathogenicity of 153 HIV-1 strains, isolated from 119 hemophiliacs, was related to the number of CD4+ lymphocytes present in the patients blood at the time of virus isolation. It was shown that the cytopathogenicity of the HIV-1 isolates was inversely correlated with the number of CD4+ lymphocytes. The highest CD4+ cell numbers were observed in 34 latently infected patients characterized by HIV seropositivity, failure of virus isolation, and detection of viral DNA by the polymerase chain reaction. Cytopathogenicity of the HIV-1 isolates was a reliable prognostic marker and correlated well with other lesssensitive prognostic parameters, including the detection of infectious virus and p24 antigen in the plasma, and the decline of p24 antibody in the serum. The results suggest that the viral isolates — if not subjected to extensive passage — represent in vivo variants selected from a heterogeneous viral population according to the particular immunological conditions of the host.

Role of HIV-1 phenotype in viral pathogenesis and its relation to viral load and CD4+ T-cell count

The predictive value of HIV‐1 phenotype in peripheral blood mononuclear cell (PBMC) coculture and the relation among viral phenotype, viral load, and CD4+ T‐cell count were examined in two studies. In study A, 132 HIV‐1–infected individuals were examined retrospectively for the relation between the result of their initial HIV cultivation in PBMC coculture and survival rate 6 years later. In study B, 176 patients were examined since 1994 for markers of HIV disease progression. HIV‐1 phenotype was determined by PBMC cocultivation, viral load by NASBA HIV RNA QT System, and CD4+ T‐cell count by flow cytometry. In study A, the percentage of survival for patients with initial negative virus culture was significantly higher (95%) than in patients with nonsyncytia‐inducing (NSI) isolates (78%) and syncytia‐inducing (SI) isolates (21%) (P < 0.05 and P < 0.0001, respectively). When SI phenotype was subdivided into moderately cytopathogenic and highly cytopathogenic, significant differences in the rate of survival between these subgroups could be observed (45% vs. 14%; P < 0.05). In study B, progression from negative virus culture to the isolation of NSI variants was associated with increasing viral load (P < 0.0001) but did not affect CD4+ T‐cell count significantly (P > 0.07), whereas the switch from NSI to SI virus was accompanied by significant decline of CD4+ T‐cells (P < 0.0001) but no change in viral load (P > 0.21). Thus, isolation and phenotyping of HIV represents an additional striking predictive marker for progression of HIV infection. J. Med. Virol. 56:259–263, 1998.

Male-to-female transmission of HIV in a cohort of hemophiliacs — Frequency, risk factors and effect of sexual counseling

SummaryThe incidence of male-to-female transmission of HIV infection was studied in a population of 198 sexual partners of hemophiliacs who tested HIV positive since 1984. The follow-up observation period was 1987–1992. Transmission occurred in 20 (10%) cases. The analysis of risk factors for transmission was performed in a subgroup of 57 hemophiliacs with seronegative sexual partners as compared to eight transmitters. Transmitters showed a significantly more advanced immune depletion at enrollment as well as at the end of the observation period. Furthermore, transmitters had a more advanced disease at the end of the study (75% vs. 29% CDC IV; p<0.01). Also virus cultures were more frequently positive in the transmitters than in the non-transmitters (71% vs. 42%). Regular sexual counseling was offered to all couples. After 1987, no new seroconversions were detected. However, two seroconversions in female partners of hemophiliacs outside the initial study population were observed. Both transmissions occurred during a period of severe clinical and immunological deterioration. This study shows that sexual partners of HIV-infected hemophiliacs with more advanced disease are at higher risk of infection with HIV. The frequency of male-to-female transmission of HIV in long-term monogamous sexual relationships practicing safer sex is low. Overall, disease awareness and counseling for safer sex seem to be effective in reducing transmission rates.ZusammenfassungDie Inzidenz der Übertragung der HIV-Infektion von Männern auf Frauen wurde in einer Population von 198 weiblichen Sexualpartnern von seit 1984 HIV-positiv getesteten Hämophilen untersucht. Die Nachbeobachtungsdauer erstreckte sich über die Jahre 1987–1992. Die Infektion wurde in 20 Fällen (10%) übertragen. Die Analyse von Risikofaktoren für die Übertragung von HIV wurde untersucht durch den Vergleich einer Untergruppe von 57 Hämophilen und seronegativen Partnern mit 8 Überträgern. Die Überträger zeigten zu Beginn als auch am Ende der Beobachtungszeit eine fortgeschrittenere Immundepletion. Darüber hinaus wiesen Überträger am Ende der Studie auch häufiger eine symptomatische Erkrankung auf (75% versus 29% CDC IV; p<0,01). Darüber hinaus war bei Überträgern 1991/92 häufiger eine positive Viruskultur nachweisbar (71% vs. 42%). Allen Paaren wurde eine regelmäßige Sexualberatung angeboten. Nach 1987 wurden keine neuen Serokonversionen beobachtet. Jedoch wurden zwei Serokonversionen bei Partnerinnen hämophiler HIV-infizierter außerhalb der ursprünglichen Population aufgedeckt. Beide Übertragungen erfolgten in einer Periode schweren klinischen und immunologischen Progresses. Unsere Studie zeigt, daß Sexualpartner von Hämophilen mit fortgeschrittener HIV-Infektion ein höheres Übertragungsrisiko aufweisen. Die Übertragungsfähigkeit von Männern auf Frauen ist auch bei langdauernden Sexualbeziehungen monogamer Paare, die safer sex praktizieren, gering. Das Wissen um das Vorliegen einer HIV-Infektion und Beratung über safer sex scheinen wirksam in der Verhütung von Neuinfektionen.

Latent human herpesvirus-6 DNA is sparsely distributed in peripheral blood lymphocytes of healthy adults and patients with lymphocytic disorders

Human herpesvirus-6 (HHV-6) can be regularly isolated from peripheral blood mononuclear cells (PBMC) of children suffering from exanthema subitum, but only rarely from PBMC of adults. Although the high prevalence of HHV-6 infection in early childhood seems to result from cell-free infectious virus shedded in saliva of healthy adults, latent HHV-6 infection is supposed to occur in lymphocytes. Therefore, we performed polymerase chain reaction (PCR) with DNA from PBMC of 44 healthy adults, 31 HIV-seropositive individuals and 33 patients with leukaemia or lymphoproliferative disorders. As positive control served PBMC from 11 children with exanthema subitum and as negative control PBMC from 20 newborns. Whereas HHV-6-specific sequences were detected in PBMC from all children with exanthema subitum and never in PBMC from newborns, they were found in PBMC of 9% of healthy adults and HIV-seropositive individuals and in 16% of the patients with lymphoproliferative disorders. Apparently detection of HHV-6 DNA in PBMC was neither limited by low sensitivity of the HHV-6 PCR assay, which detected less than ten copies of cloned HHV-6 DNA, nor by a low rate of latently infected individuals, but was limited by the number of lymphocytes subjected to PCR. It is supposed that the presence of latent HHV-6 DNA in lymphocytes is common, but that infected lymphocytes are rare (≤1 infected cell in 105 lymphocytes).Although the results do not support a definite propagation of HHV-6 in HIV-seropositive individuals or in patients with leukaemia or lymphoproliferative disorders, they cannot exclude HHV-6 acting as co-factor in HIV infection or in lymphoproliferative disorders.

Different Mechanisms of Protection by Monoclonal and Polyclonal Antibodies during the Course of Herpes Simplex Virus Infection

Young adult C57BL mice intravaginally inoculated with herpes simplex virus type 1 and passively immunized with a monoclonal antibody directed against herpes simplex virus type 1 glycoprotein gB were shown to be more effectively protected against infection as compared with mice treated with polyclonal immune serum. In contrast to polyclonal antiserum, the monoclonal antibody markedly restricted viral multiplication in the infected mucous membranes. Consequently, skin lesions were completely prevented, and the extent of ganglionic infection was significantly reduced. The mechanism by which a monoclonal antibody, specific to glycoprotein gC, effected protection also differed from that of the hyperimmune serum, since premature latency was not induced. The data provide strong evidence that the mechanisms of protection mediated by antibodies depend on their epitope specificity. The inhibition of active antibody response after passive immunization was inducible by polyclonal antibody only, not by monoclonal antibodies.

Detection of latent thymidine kinase-deficient herpes simplex virus in trigeminal ganglia of mice using the polymerase chain reaction

SummaryLatency of thymidine kinase-negative mutants of herpes simplex virus (TK− HSV) could not be detected by reactivating the virus from the ganglia of infected mice. Because Southern blot hybridization was not sensitive enough to detect viral DNA, positive results obtained by dot blot hybridization were ascertained by the highly specific and sensitive polymerase chain reaction (PCR), which detected both latent TK− HSV type 1 and 2 DNA from the trigeminal ganglia of infected mice.

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity

Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4+ and CD8+ cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351–360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379–385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.