Lacked Neutralizing Activity Research Articles

Virological and immunological correlates of HIV post-treatment control after temporal antiretroviral therapy during acute HIV infection.

People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary post-treatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T-cells and HIV-specific T-cell responses were analyzed by flow cytometry. The case presented with AHI and a plasma viral load of 2.7 million copies/mL. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption HIV-DNA in CD4+ T-cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8+ T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44:02 restricted gag-epitope, which was associated with decreased replication. We describe an exceptional case of post-treatment control, which is likely associated with sustained potent gag-specific CD8+ T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.

Sotrovimab in Hospitalized Patients with SARS-CoV-2 Omicron Variant Infection: a Propensity Score-Matched Retrospective Cohort Study.

In vitro data suggest the monoclonal antibody sotrovimab may have lost inhibitory capability against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. We aimed to provide real-life data on clinical outcomes in hospitalized patients. We retrospectively analyzed patients who were treated at the University Medical Center Hamburg-Eppendorf, Germany, between December 2021 and June 2022. Out of all 1,254 patients, 185 were treated with sotrovimab: 147 patients received sotrovimab monotherapy, and 38 received combination treatment with sotrovimab and remdesivir. We compared in-hospital mortality for the different treatment regimens for patients treated on regular wards and the intensive care unit separately and performed propensity score matching by age, sex, comorbidities, immunosuppression, and additional dexamethasone treatment to select patients who did not receive antiviral treatment for comparison. No difference in in-hospital mortality was observed between any of the treatment groups and the respective control groups. These findings underline that sotrovimab adds no clinical benefit for hospitalized patients with SARS-CoV-2 Omicron variant infections. IMPORTANCE This study shows that among hospitalized patients with SARS-CoV-2 Omicron variant infection at risk of disease progression, treatment with sotrovimab alone or in combination with remdesivir did not decrease in-hospital mortality. These real-world clinical findings in combination with previous in vitro data about lacking neutralizing activity of sotrovimab against SARS-CoV-2 Omicron variant do not support sotrovimab as a treatment option in these patients.

Cross-Protective Immune Responses Induced by Sequential Influenza Virus Infection and by Sequential Vaccination With Inactivated Influenza Vaccines.

Sequential infection with antigenically distinct influenza viruses induces cross-protective immune responses against heterologous virus strains in animal models. Here we investigated whether sequential immunization with antigenically distinct influenza vaccines can also provide cross-protection. To this end, we compared immune responses and protective potential against challenge with A(H1N1)pdm09 in mice infected sequentially with seasonal A(H1N1) virus followed by A(H3N2) virus or immunized sequentially with whole inactivated virus (WIV) or subunit (SU) vaccine derived from these viruses. Sequential infection provided solid cross-protection against A(H1N1)pdm09 infection while sequential vaccination with WIV, though not capable of preventing weight loss upon infection completely, protected the mice from reaching the humane endpoint. In contrast, sequential SU vaccination did not prevent rapid and extensive weight loss. Protection correlated with levels of cross-reactive but non-neutralizing antibodies of the IgG2a subclass, general increase of memory T cells and induction of influenza-specific CD4+ and CD8+ T cells. Adoptive serum transfer experiments revealed that despite lacking neutralizing activity, serum antibodies induced by sequential infection protected mice from weight loss and vigorous virus growth in the lungs upon A(H1N1)pdm09 virus challenge. Antibodies induced by WIV vaccination alleviated symptoms but could not control virus growth in the lung. Depletion of T cells prior to challenge revealed that CD8+ T cells, but not CD4+ T cells, contributed to cross-protection. These results imply that sequential immunization with WIV but not SU derived from antigenically distinct viruses could alleviate the severity of infection caused by a pandemic and may improve protection to unpredictable seasonal infection.

Porcine reproductive and respiratory syndrome virus neutralizing antibodies provide in vivo cross-protection to PRRSV1 and PRRSV2 viral challenge

Vaccine control and prevention of porcine reproductive and respiratory syndrome (PRRS), the most important disease of swine, is difficult to achieve. However, the discovery of broadly neutralizing antibody activity against porcine reproductive and respiratory syndrome virus (PRRSV) under typical field conditions opens the door to new immunologic approaches for robust protection. We show here that passive administration of purified immunoglobulins with neutralizing antibodies reduced PRRSV2 infection by up to 96%, and PRRSV1 infection by up to 87%, whereas immune immunoglobulins lacking neutralizing activity had no effect on viral infection. Hence, immune competence of passive immunoglobulin transfer was associated specifically with antibody neutralizing activity. Current models of PRRSV infection implicate a minor envelope glycoprotein (GP) complex including GP2, GP3, and GP4, as critical to permissive cell infection. However, conserved peptides comprising the putative cell attachment structure did not attenuate neutralization or viral infection. The results show that immunological approaches aimed at induction of broadly neutralizing antibodies may substantially enhance immune protection against PRRSV. The findings further show that naturally occurring viral isolates are able to induce protective humoral immunity against unrelated PRRSV challenge, thus removing a major conceptual barrier to vaccine development.

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.

Characterization of antibody response in neuroinvasive infection caused by Toscana virus

ObjectivesAmong sandfly-borne pathogens, Toscana virus (TOSV) is a prominent cause of summer meningitis in Mediterranean Europe. Here, we assessed the kinetics of anti-TOSV antibodies over time in 41 patients diagnosed with TOSV meningitis or meningoencephalitis in northeastern Italy. MethodsAcute and follow-up serum samples were collected up to 20 months after diagnosis of TOSV infection and tested for the presence of specific antibody using immunoenzymatic and indirect immunofluorescence assays. In addition, maturation of anti-TOSV IgG over time was evaluated as well as production of neutralizing antibodies. ResultsSpecific IgM and IgG response was present at diagnosis in 100% of patients; TOSV-specific IgM and IgG were detected in patients' sera up to 6 and 20 months after diagnosis, respectively. The avidity index (AI) increased over the first month after infection in 100% of patients and most cases exceeded 60% by Day 30 post infection. The AI subsequently plateaued then declined at 20 months after diagnosis. Finally, neutralization assay to TOSV was performed in 217 sera collected from 41 patients; 69.6% of tested samples resulted in reactive and moderate levels of neutralizing antibodies observed during all phases of infection despite high titres of total anti-TOSV IgG. ConclusionsSpecific antibody response develops rapidly and is long-lasting for neuroinvasive TOSV infection. Serodiagnosis of neuroinvasive TOSV requires simultaneous detection of specific IgM and IgG. Moderate levels of neutralizing antibodies were maintained over the study period, while the protective role of antibodies lacking neutralizing activity is unclear and requires further evaluation.

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 (671NWFDITNWLWYIK683). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols.

Severe group A streptococcal infections in Uppsala County, Sweden: Clinical and molecular characterization of a case cluster from 2006 to 2007

This study describes a recent cluster of 30 patients (median age 52 years) with serious group A streptococcal (GAS) infections in Uppsala County, Sweden, from December 2006 to May 2007. Patients hospitalized with a severe GAS infection, i.e. cases with either invasive GAS (iGAS) disease or patients with a positive non-sterile site culture/rapid antigen test for GAS and clinically considered as having a critical disease, were included in the study. Common clinical presentations were skin and soft tissue infections (53%) and pneumonia (17%). Eight patients (27%) were diagnosed with streptococcal toxic shock syndrome. In 40% of the cases no relevant underlying disease was reported. Among the 16 patients with soft tissue infections, the upper chest, neck or upper arm area was frequently affected and the infection was associated with severe pain. Among the 20 collected isolates, the T1/emm1 type dominated (80%). The majority (86%) of 7 analysed acute sera lacked neutralizing activity against superantigens produced by the patients' own infecting isolate. The study underscores the association between T1/emm1 and outbreaks of serious GAS infections. This highlights the importance of surveillance for prompt identification of more aggressive isolates in the community, thereby increasing awareness among healthcare professionals of these life-threatening infections.

Enhanced Immunity and Protective Efficacy against SIVmac251Intrarectal Challenge Following Ad-SIV Priming by Multiple Mucosal Routes and gp120 Boosting in MPL-SE

Previously, 39% of rhesus macaques primed orally, intranasally, and intratracheally with adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinants and boosted with gp120 in monophosphoryl lipid A-stable emulsion (MPL-SE) remained aviremic or cleared or controlled viremia at the threshold of detection following SIV(mac251) intrarectal challenge (Study B). In contrast, no macaques primed orally and intranasally with Ad-SIV recombinants and boosted with gp120 in Quillaja Saponaria-21 exhibited undetectable viremia post-challenge (Study A). We conducted a detailed comparison of the studies to elucidate the effect of different vaccine regimens on induced immunity associated with the different challenge outcomes. Quantitative viral load comparisons were statistically analyzed. All immune responses were assessed at identical timepoints post-immunization, and cellular immunity was re-evaluated on cryopreserved cells from Study B macaques to match Study A data acquired with frozen cells. Study B exhibited greater protective efficacy, increased levels of p11C and p54m tetramer positive cells and a trend toward enhanced interferon-gamma secreting cells in response to Env and Gag peptides, modestly enhanced serum neutralizing antibodies, and greater positivity in anti-gp120 rectal IgA and IgG antibodies. Study A macaques exhibited greater positivity in salivary IgA anti-gp120 antibodies. Thus, the vaccine regimen using oral-intranasal-intratracheal priming and protein boosting in MPL-SE was superior, eliciting greater protective efficacy against pathogenic SIV(mac251) and enhanced SIV-specific immunity, systemically and at rectal sites. The mechanism(s) by which binding antibodies, lacking neutralizing activity against the primary challenge virus, may contribute to protection requires further study.

The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus

I have used indirect ELISA with overlapping synthetic peptides representing the GP5 ectodomain to study the generation and specificity of peptide-binding Abs in pigs that were infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) strain VR2332 and in North American field sera submitted for PRRSV infection diagnosis. Peptide-binding Abs appeared in sera of the VR2332-infected pigs within about 30 days post-farrowing (dpf), reaching maximum titers 100-200 dpf and then decreasing slowly to about half of maximum titer by about 400 dpf. The formation of peptide-binding Abs and of virus neutralizing Abs correlated and their initial appearance coincided with disappearance of virus from the circulation. The Abs were specific for VR2332-specific peptides. In contrast, anti-N-protein Abs as measured by HerdCheck ELISA appeared within 7 dpf, reached maximum levels at about 100 dpf and had decreased below detectable levels by about 200 dpf. Twenty-seven field serum samples with virus neutralizing activity all possessed high levels of peptide binding Abs, but the Abs bound about equally to VR2332 and strain Lelystad virus (LV)-specific peptides. The indirect ELISA results using various large peptides and competition ELISA using small peptides (8 or 9 amino acids long) confirmed that the epitope recognized by the Abs is located in the GP5 ectodomain sequence 37SHLQLIYNL of VR2332. Use of mutated peptides in the competition ELISA showed that 42I to T and 38HL to TY substitutions blocked Ab recognition, whereas deletion of 41L had no effect. In addition, 26 serum samples submitted by two farms for diagnostic tests were found to possess low levels of Abs that bound to GP5 ectodomain peptides, even though the sera were sero-negative in the HerdChek ELISA and lacked neutralizing activity. Competition ELISA showed that the Abs recognized one or more epitopes located downstream of the PRRSV neutralization epitope. An epitope(s) located in the same area was recognized by Abs generated in mice by immunization with a GP5 ectodomain peptide conjugated to BSA. These Abs also lacked neutralizing activity.

Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects.

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Safety and Efficacy of Saquinavir Soft-Gelatin Capsules + Zidovudine + Optional Lamivudine in Pregnancy and Prevention of Vertical HIV Transmission

Summary: There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant crossclade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Lack of Autologous Neutralizing Antibodies in the Cerebrospinal Fluid of HIV-1 Infected Individuals

The cerebrospinal fluids (CSF) and sera from HIV-1-infected individuals at different clinical stages were monitored for neutralizing activity against CSF-derived HIV-1 isolates. None of the CSF samples and only one of seven serum samples could neutralize the autologous CSF isolate. CSF samples collected one to two years later from the same patients also lacked autologous neutralizing antibodies against these isolates. However, some CSF samples were able to neutralize heterologous CSF isolates albeit in low titers. HIV antibody positive control sera could readily neutralize all of the CSF isolates demonstrating that these isolates were not resistant to neutralization per se. IgG antibodies against the HIV-1 envelope protein and, specifically, against the V3 loop of HIV-1 gp120 (MN) were present in some CSF samples, although the samples lacked neutralizing activity. In summary, this study demonstrates a lack of autologous neutralizing antibodies in CSF samples when assayed against CSF-derived HIV-1 isolates.

Antigenic and Functional Characterization Of Rinderpest Virus Envelope Proteins Using Monoclonal Antibodies

A total of 24 monoclonal antibodies (MAbs) against the haemagglutinin (H) and the fusion protein (F) of rinderpest virus (RPV) were used to characterize their antigenic structure and biological properties, and to analyse natural variation in the envelope proteins of morbilliviruses. The anti-H and anti-F MAbs defined seven and three distinct antigenic sites, respectively. The MAbs to six sites on H were able to neutralize the infectivity of RPV. The addition of guinea-pig complement or anti-mouse immunoglobulin increased the virus-neutralizing antibody titre of most of the anti-H MAbs, including those lacking neutralizing activity. One of the antigenic sites on H was conserved among morbilliviruses and the MAbs to this site had haemagglutination inhibition activity against measles virus (MV). The remaining sites were specific for RPV and varied antigenically between strains of RPV. The anti-F MAbs lacked neutralizing activity, but two of the five MAbs did show activity in the presence of complement or anti-mouse immunoglobulin. On the whole, the antigenic sites on F were conserved in some strains of MV, but not in canine distemper virus. All of the sites on the surface proteins were sensitive to SDS and, although those on F were not affected by 2-mercaptoethanol, five of the seven sites on H were destroyed by it. These results suggest that the epitopes on the envelope proteins are conformation-dependent.

A recombinant vaccinia virus expressing the major capsid protein of Simian rotavirus-induced anti-rotavirus antibodies

cDNA molecules encoding the major structural protein (VP6) of the Simian rotavirus SA11 were inserted under the control of the vaccinia virus 7.5 kDa promoter into the thymidine kinase gene. Synthesis of VP6 was demonstrated by immunoprecipitation of recombinant virus-infected cell. Mice inoculated via several routes with this recombinant vaccinia produce high titers of antirotavirus antibodies lacking neutralizing activity.

Relationship between HTLV-III neutralizing antibody and clinical status of pediatric acquired immunodeficiency syndrome (AIDS) and AIDS-related complex cases.

To investigate a possible protective role of HTLV-III neutralizing antibodies in individuals exposed to the virus, sera of children with acquired immunodeficiency syndrome or acquired immunodeficiency syndrome-related complex were analyzed for neutralizability of HTLV-IIIB infectivity. Twelve pediatric patients (nine acquired immunodeficiency syndrome, three acquired immunodeficiency syndrome-related complex) were clinically stable and had survived more than 2 yr postonset. Their predominant clinical problems included lymphocytic interstitial pneumonia, candidiasis, recurrent bacterial infections, failure to thrive, and lymphadenopathy. Twelve additional children (all acquired immunodeficiency syndrome) were classified as clinically poor; 10 of them had died. Their median length of survival was less than 2 yr, and their disease spectrum included progressive encephalopathy, thymic depletion or atrophy, and Pneumocystis carinii pneumonia in addition to many of the clinical features of the stable children. All (100%) of the stable patients possessed serum neutralizing antibody in contrast to only one of the 12 (8%) clinically poor patients. A simple decline in immunologic reactivity to HTLV-III antigens with disease progression did not account for this difference, since HTLV-III antibody titers of the clinically poor cases (1 X 10(2) to 1 X 10(7)) ranged as high as those of the stable cases (1 X 10(4) to 1 X 10(7)) when measured by the ELISA technique. Although stable cases possessed a higher geometric mean titer (2.8 X 10(5)) by ELISA than the poor cases (4 X 10(4)), this difference was not statistically significant. Serial serum samples from stable children exhibited continual neutralizing antibody activity while two of three clinically poor cases lacked neutralizing activity in serial specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroblast growth enhancing activity of tumor necrosis factor and its relationship to other polypeptide growth factors.

Tumor necrosis factor (TNF) is a monocyte-derived protein cytotoxic or cytostatic for some tumor cell lines. Here we show that highly purified E. coli-derived recombinant human TNF stimulated the growth of human FS-4 diploid fibroblasts. Stimulation of cell growth was demonstrable at a TNF concentration of 10 pg/ml (3 X 10(-13) M). Maximal stimulation was attained at TNF concentrations of 10 ng/ml (3 X 10(-10) M) or higher. Growth-stimulatory activity of TNF was inhibited by an mAb neutralizing the cytotoxic activity of TNF. Growth stimulation was not inhibited by another mAb specific for TNF, lacking neutralizing activity for the cytotoxic activity of TNF. Growth stimulation by TNF was more marked and more sustained in the presence of greater than or equal to 10% FCS than in medium with less than or equal to 5% FCS. Addition of TNF to confluent FS-4 cultures also produced a marked stimulation of cell growth in the presence of fresh FCS, while a much less marked stimulation was seen in the absence of FCS. Stimulation of confluent cultures by TNF in serum-free medium was enhanced by insulin, suggesting that insulin or insulin-like growth factor(s) in the serum can act synergistically with TNF in producing growth stimulation. While the growth-stimulatory effects of TNF and insulin were synergistic, the actions of TNF and epidermal growth factor (EGF) were less than additive, suggesting that TNF and EGF may activate identical or similar pathways. We conclude that stimulation of cell growth is probably a physiological function of TNF, and that the cytotoxic and cytostatic actions of TNF may be the result of an anomalous growth signal transduction in neoplastic cells lacking the constraints of normal growth control mechanisms.

Varicella-zoster viral glycoproteins analyzed with monoclonal antibodies.

Monoclonal antibodies to varicella-zoster virus were used to study viral glycoproteins by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on the viral glycoproteins immunoprecipitated, the five monoclonal antibodies fell into three groups. Two antibodies, 4B7 and 8G9 (group 1), immunoprecipitated a single glycoprotein of molecular weight (MW) 118,000 (118K glycoprotein) and had high neutralizing activity in the absence of complement. One antibody, 3C7 (group 2), which lacked neutralizing activity, immunoprecipitated two glycoproteins of MWs 120,000 and 118,000 and a glycoprotein giving a diffuse band in the region of 64,000 to 65,000. Pulse-chase experiments and experiments with monensin as an inhibitor of glycosylation suggested that the 120K polypeptide was derived by glycosylation of the 118K polypeptide and that a 43K antigen was processed into the 64 to 65K glycoprotein. Two antibodies, 3G8 and 4E6 (group 3), both had neutralizing activity only in the presence of complement, and both immunoprecipitated at least five polypeptides, with MWs ranging from 50,000 to 90,000. Antibody 3G8 was isotype immunoglobulin G2b (IgG2b), and its immunoprecipitating activity was stronger than that of 4E6, which was isotype IgG1. Pulse-chase experiments with antibody 3G8 showed that lower-MW glycopeptides chased into three polypeptides of MWs 90,000, 80,000, and 60,000 by 24 h. Immunoprecipitation experiments with antibody 3G8 on infected cells treated with glycosylation inhibitors 2-deoxyglucose, monensin, and tunicamycin, suggested that a prominent, early-appearing 70K polypeptide may have been processed into the glycoproteins of higher MWs and that the 60K polypeptide may have been derived by glycosylation of polypeptides of lower MWs.

The interaction of monoclonal antibodies directed against envelope glycoprotein E 1 of Sindbis virus with virus-infected cells

Two monoclonal antibodies specific for the Sindbis virus envelope glycoprotein E 1 were evaluated for their ability to maintain long-term infection when present in the medium of virus-infected cells. One of them, previously shown to have neutralizing activity and to inhibit haemagglutination, caused suppression of both virus expression at the cell surface and prolonged intracellular virus presence. The other monoclonal antibody which lacked neutralizing activity but inhibited virus-specific haemolysis caused redistribution of viral antigens on the cell surface but only slightly prolonged cell survival. Both epitopes were located on the surface of the virus. By electron microscopy it was demonstrated that the determinant associated with haemolytic activity resided near the virus membrane while the haemagglutination inhibition antibody attached near the apex of the virus spikes.