Measurement Of Neutralizing Antibodies Research Articles

Interferon-β1b for multiple sclerosis

Interferon-β1b (Betaseron®/Betaferon®) was the first approved therapy for relapsing-remitting multiple sclerosis. The US Food and Drug Administration has expanded the indication to include relapsing forms of multiple sclerosis which encompasses secondary-progressive multiple sclerosis if relapses are present. In one scientifically sound head-to-head comparison (Independent Comparison of Interferon trial), interferon-β1b was shown to be clinically superior to low-dose interferon-β1a (Avonex™). Current studies are underway to compare it with a double dosage of interferon-β1b as well as glatiramer acetate. Neutralizing antibodies are more likely to occur with interferon-β1b, but their clinical significance has shown conflicting and confusing results making the utility of measuring neutralizing antibodies uncertain. Up to 12 years of follow-up data suggest that the drug remains effective on T2 magnetic resonance imaging burden of disease in those who stay on therapy. Initially, the major problem with interferon-β1b was a lack of tolerability due to high incidents of skin reactions and influenza-like side effects. Patient adherence has improved dramatically with the introduction of autoinjectors and protocol changes including initial dose escalation, prophylactic ibuprofen or acetaminophen, evening administration of drug and an attentive nurse support system. Interferon-β1b remains a first-line treatment for relapsing-remitting multiple sclerosis and relapsing forms of secondary-progressive multiple sclerosis based on robust efficacy data and a long-term safety profile.

Immunogenicity of baculovirus expressed recombinant proteins of Japanese encephalitis virus in mice

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E+prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.

A comparative evaluation of nasal and parenteral vaccine adjuvants to elicit systemic and mucosal HIV-1 peptide-specific humoral immune responses in cynomolgus macaques

Cynomolgus macaques were immunized by either the intramuscular (i.m.) or intranasal (i.n.) route with a HIV-1 peptide-based immunogen (C4-V3 89.6P) alone, or formulated with novel adjuvants to evaluate the ability of the adjuvants to augment peptide-specific systemic and mucosal immune responses. A mutant cholera toxin, CT-E29H, or the combination of recombinant human IL-1α (rhIL-1α) protein and recombinant human GM-CSF (rhGM-CSF) protein were tested as adjuvants for i.n. immunization, while a stable emulsion of a synthetic monophosphoryl lipid A (MPL) analogue (RC529-SE) plus rhGM-CSF protein was tested as an adjuvant for i.m. immunization. Macaques immunized i.n. with peptide alone failed to elicit an anti-C4-V3 89.6P antibody response in serum. In contrast, all the tested peptide/adjuvant formulations elicited peptide-specific immune responses. RC529-SE/rhGM-CSF elicited the highest peak anti-peptide IgG geometric mean titer in serum (1:32,768 at week 25) followed by rhIL-1α/rhGM-CSF (1:1217 at week 10) and CT-E29H (1:256 at week 25). Measurable SHIV neutralizing antibody responses were detectable in only one macaque immunized i.m. with peptide formulated with RC529-SE/rhGM-CSF. Macaques immunized by the i.n. route with peptide in combination with CT-E29H failed to elicit measurable antibody responses at nasal or genital mucosal surfaces. In contrast, antibody responses at the nasal and genital mucosa were detected in macaques immunized by the i.n. route with peptide in combination with rhIL-1α/rhGM-CSF. However, antibody responses at the nasal and genital mucosa were highest in macaques immunized parenterally with peptide in combination with the adjuvants RC529-SE/rhGM-CSF. These results suggest that parenteral vaccine administration in combination with the appropriate adjuvant formulation can elicit vaccine-specific humoral immune responses in both systemic and mucosal compartments.

Antibody-mediated protection against cytotoxic T-cell escape in coronavirus-induced demyelination.

C57BL/6 (B6) mice infected with mouse hepatitis virus (MHV) strain JHM develop a clinically evident, demyelinating encephalomyelitis. Infectious virus can be isolated from the spinal cords of these mice and is invariably mutated in the immunodominant CD8 T-cell epitope recognized in this strain. We showed previously that these persistently infected mice did not mount a measurable serum anti-MHV neutralizing antibody response. Here we show that cytotoxic T-lymphocyte (CTL) escape was not detected in MHV-infected BALB/b mice (H-2(b) haplotype), even though the same CD8 T-cell epitopes were recognized as in B6 mice. BALB/b mice had 25-fold more MHV-specific antibody-secreting cells in the central nervous system, the site of infection, than B6 mice, suggesting that local production of anti-MHV antibody contributed to this absence of CTL escape. Additionally, administration of anti-MHV neutralizing antibody to infected B6 mice suppressed the development of CTL escape mutants. These findings indicate a key role for the anti-MHV antibody response in suppressing virus replication, thereby minimizing the emergence and competitive advantage of CTL escape mutants.

Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

Echovirus 4 and type 1 Diabetes Mellitus

Aims/hypothesis: To determine the association between exposure to enteroviruses and Type I diabetes. Methods: We measured neutralizing antibodies to the following enteroviruses: Coxsackievirus CA9, CB1, CB2, CB3, CB4, CB5, CB6, and Echovirus E4, E6, E9, El 1 in the sera of (1) Type I diabetic patients at diagnosis (n = 33), (2) healthy offspring of parents with Type I diabetes without islet cell antibodies (ICA) (n = 43) and (3) normal controls (n = 57). All subjects were less than 20 years old. We performed the neutralization test determining the cytopathogenic effect on Vero cells. HLA DR serotyping was also performed in Group 2. Results: Type I diabetic patients showed a higher frequency (21.2%, p < 0.01) of neutralizing antibodies to E4 in relation to controls (1.8%), although there were no differences comparing with offspring of Type 1 diabetic patients (20.9%). Healthy offspring carrying Type I diabetes HLA DR susceptibility genes were also exposed to E4 (15.0%). High frequencies of neutralizing antibodies to most enteroviruses were found in the control group. Conclusion: This study shows the association between Type I diabetes and the presence of neutralizing antibodies to Echovirus 4, suggesting the possible participation of this virus as an environmental trigger of this autoimmune disease. Interestingly, our population displays high frequencies of exposure to enterovirus (including CB4) although the incidence for Type 1 diabetes is low (2.9 per 100,000 inhabitants).

Lassa fever: implications of T-cell immunity for vaccine development

Lassa fever is a re-emerging viral hemorrhagic fever, which causes significant human morbidity in endemic regions of West Africa. Attempts to vaccinate against this virus in animal models including non-human primates have revealed that eliciting a strong cellular immune response protects from clinical disease, but not infection, in the absence of measurable neutralizing antibodies. As there is renewed interest in developing a vaccine against Lassa fever for use in humans, several questions should be addressed in view of the scarce knowledge of the mechanisms of natural immunity against this disease. MHC-dependency of a vaccine relying mainly on the induction of T-cell immunity and its ability to cross-protect against different Lassa virus strains will be important issues. Furthermore, the question whether the vaccine can prevent human-to-human transmission of the virus should be discussed and the possibility that vaccination could predispose to immunopathology should be excluded. We are addressing some of the above mentioned problems concerning natural immunity through field studies in the Republic of Guinea, West Africa, and are presently studying the CD4 cell responses of Lassa antibody positive subjects on the basis of T-cell proliferation assays using recombinant Lassa virus proteins.

Env-independent protection induced by live, attenuated simian immunodeficiency virus vaccines.

Live attenuated simian immunodeficiency viruses (SIV), such as nef deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a nef deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Uberla, J. Virol. 71:2225-2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the nef deletion mutant SIVDeltaNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVDeltaNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the env gene of SIV had been functionally replaced by the env gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.

Production of Reassortant Viruses Containing Human Rotavirus VP4 and SA11 VP7 for Measuring Neutralizing Antibody following Natural Infection

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.

Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection.

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.

Characterization of a CD4-Expressing Macaque Cell Line That Can Detect Virus after a Single Replication Cycle and Can Be Infected by Diverse Simian Immunodeficiency Virus Isolates

Primate lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are phenotypically diverse, and virus isolates vary in cytopathicity, replication rate, and cell tropism. While all virus isolates infect primary peripheral blood lymphocytes, only a subset of strains infect established CD4-expressing T-cell lines. Here, we describe the development and characterization of a macaque cell line that can be infected by all of the strains of SIV that we have tested, including macrophage- and T-cell-tropic strains, primary and cell-line adapted strains, and SIVmac, SIVMne, and SIVsm isolates. The cells can be infected by strains of HIV type 2 (HIV-2) to varying degrees, but not by either cloned or primary isolates of HIV type 1 (HIV-1). This cell line is a derivative of a rhesus macaque mammary tumor cell line (CMMT) engineered to express human CD4. For these studies, a CMMT-CD4 clone expressing an integrated copy of a truncated HIV-1 long terminal repeat fused to theβ-galactosidasegene (LTR-β-gal) was established to allow detection of infectious SIV after a single round of replication. Here, we demonstrate the ability of the CMMT-CD4-LTR-β-galcell line to rapidly and quantitatively detect infectious SIV. Using these cells to assay virus, we could readily measure neutralizing antibody activity in animals infected with different SIV isolates. Neutralizing activity was detected against the homologous virus and lower, but detectable, activity was measured against heterologous virus. Thus, this system, which is highly sensitive and can detect infection by all of the SIV isolates we tested, is a rapid method for detecting infectious virus and quantitating neutralizing antibody activity.

Neither Whole Inactivated Virus Immunogen nor Passive Immunoglobulin Transfer Protects Against SIVagm Infection in the African Green Monkey Natural Host

Attempts to protect against infection with simian immunodeficiency virus (SIV)mac grown in rhesus peripheral blood mononuclear cells (PBMCs) using whole inactivated virus immunogen or passive transfer of antibody have so far universally failed. However, such experiments have succeeded in the closely related human immunodeficiency virus (HIV)-2/cynomolgus system. To determine whether the failure in the SIVmac system is typical of primate lentiviruses we performed vaccination and passive transfer experiments using SIVagm (genetically distinct from SIVmac and HIV-2) in the natural African green monkey (AGM) host. To maximize the chances of success, both immunogen and challenge material were prepared using the molecular SIVagm3 clone. Two AGMs were immunized four times with whole inactivated SIVagm3 in RIBI adjuvant and two received intravenously a high dose of immunoglobulin (Ig) purified from a mixture of plasma from AGM, either naturally infected or infected with the homologous SIVagm3 clone. All were challenged with 20 median minimum infective doses of the SIVagm3 grown in AGM PBMCs and previously titrated in AGMs. Despite a strong humoral immune response in all monkeys at the time of challenge, including measurable neutralizing and antibody-dependent cell-mediated cytotoxicity antibodies, none were protected from infection.

Antibody response to 146S particle, 12S protein subunit and isolated VP1 polypeptide of foot-and-mouth disease virus type Asia-1

The antibody response to foot-and-mouth disease virus (FMDV) antigens of type Asia-1 in guinea-pigs was studied by micro-serum neutralization test (MSNT) and enzyme-linked immunosorbent assay (ELISA). One inoculation of as little as 1 μg of binary ethyleneimine (BEI)-insctivated 146S virus particles in guinea-pigs elicited enough neutralizing antibodies to protect them against challenge with virulent virus. However, one inoculation of live 146S virus particles elicited higher levels of neutralizing antibodies in guinea-pigs than that of inactivated 146S particles. One inoculation of 12S protein subunits in guinea-pigs elicited only non-neutralizing anti-12S antibodies detected by ELISA. Similarly, non-neutralizing anti-VP1 antibodies were detected by ELISA after one inoculation of VP1. However, multiple inoculations of 12S or VP1 elicited measurable neutralizing antibodies in guinea-pigs that protected them against challenge.

Antibody response of kittens after vaccination followed by exposure to feline leukemia virus-infected cats

Summary Protein (western) blot analysis and virus-neutralization assay were used to evaluate the antibody response of specific-pathogen-free kittens to FeLV vaccination and followed by natural exposure. Several kittens had barely detectable reactions to specific FeLV antigens prior to vaccination or exposure. Correlation was not found between protection against persistent viremia and antibody response after vaccination as measured by western blot analysis or virus neutralization assay. A statistically significant (P &lt; 0.01) difference in the antibody response against p27 antigen after natural exposure to FeLV was observed between persistently viremic kittens and transiently viremic or aviremic kittens. Measurable (P &lt; 0.05) virus neutralizing antibody titer after FeLV exposure was found only in a small number of kittens that were protected against persistent viremia. Lack of association between humoral response and vaccination-induced protection against persistent FeLV infection suggests an important role for cell-mediated immunity in such protection.

Development of an improved method for measuring neutralizing antibody to rotavirus

An improved assay was developed for measuring neutralizing antibody titers against rotaviruses. This procedure used the same initial steps as performed to determine antibody titers by the focus reduction neutralization (FRN) assay. However, instead of counting infected cells after staining with fluorescein, reductions in virus infectivity by neutralizing antibody were determined by quantitation of viral antigen production using an ELISA. A linear relationship was found between ELISA absorbance values and focus forming units for each of four prototype rotaviruses, representative of serotypes 1–4. Thus, the serum dilution that resulted in neutralization of 60% of infectious virus (i.e. the neutralizing antibody titer) could be readily determined from absorbance values. Titers found by this method were similar to and as reproducible as those found by the FRN assay. Because this method is less laborious and the results are obtained by objective rather than subjective methods, it represents an improvement over the FRN assay.

A Simple Antigen-Reduction Assay for the Measurement of Neutralizing Antibodies to Hepatitis A Virus

A simplified hepatitis A virus (HAV) antigen-reduction neutralization assay (HAVARNA) was developed to permit the measurement of biologically active antibodies in recipients of candidate HAV vaccines. Degrees of neutralization were measured from the reduction in the amount of HAV antigen synthesized by 7-10 days after infection of MRC-5 (fetal human diploid lung) cell cultures. Sera producing a greater than or equal to 50% reduction in viral infectivity were scored as neutralizing. The assay was applied to demonstrate serum HAV neutralizing activity in 10 of 10 and 9 of 10 recipients of 10(7) and 10(6) TCID50 doses, respectively, of the Merck CR326F (F' variant) live attenuated vaccine. The dilution end points of selected sera ranged from 1:10 to 1:640. The dilution end point of the World Health Organization reference globulin no. 1 was 1:530,000 (0.2 mlU/ml of HAV antibody). The HAVARNA provided a rapid, sensitive, and reproducible means to measure neutralizing antibodies to HAV.

A sensitive method for measuring neutralizing antibodies to Bordetella pertussis toxin: Optimized ADP-ribosylation of transducin

Pertussis toxin (PT), preactivated with 20 m m dithiothreitol (DTT), was incubated with different serum dilutions ( 1 10 – 1 200 ) before addition to the reaction mixture. Final concentrations of the reagents were: PT (50 ng/ml), dithiothreitol (DTT)(≤ 0·3 m m), bovine transducin (2 μg/ml), ATP (1 m m). GTP (1 m m), lysophosphatidylcholine (LPC) (0·1 mg/ml), sodium acetate (NaAc) (0·1 m), Tris-HCl, pH 7·1 (0·06 m) and 32P-NAD + (10 μCi, 28 Ci/m m). The reaction was stopped by precipitation with 10% TCA ( w v ), the pellet was collected and the samples were submitted to SDS-PAGE followed by autoradiography. ADP-ribosylation was detected as the radiolabelling of a protein band (m.w. ≈ 40 kD) corresponding to the α-subunit of transducin. 32P-ADP incorporation was a linear function of PT concentration. By this assay quantitative differences in PT neutralizing antibodies were found between sera which were not revealed by measuring PT neutralizing antibodies by a Chinese hamster ovary (CHO) cell culture test or by an enzyme-linked immunosorbent assay (ELISA). The conditions of the ADP-ribosylation of bovine transducin have been optimized to permit detection of the enzymic activity of as low amounts of PT as 0·5 ng. This opens the possibility of the study of the presence and avidity of neutralizing antibodies to PT in post-vaccination sera without preceding purification and concentration of the antibodies and thus may provide a useful tool for evaluation of whooping cough vaccines.

A refined complement-enhanced neutralization test for detecting antibodies to Junin virus

A refined, complement-enhanced, plaque-reduction neutralization test was developed for measuring neutralizing antibodies against Junin (Argentine hemorrhagic fever) virus. The assay measured neutralizing antibodies after natural as well as vaccine-induced Junin virus infections. Among vaccinated individuals, titers were 2–4-fold higher than those obtained with conventional assays, without loss of specificity. Enhanced sensitivity was achieved by using a standardized complement source (vs human or animal serum) for virus dilution, incubation of virus-serum mixtures at 36°C for 2 h (vs overnight at 4°C) prior to plaque assay, control of age and density of cell monolayers, and variation in overlay conditions.

A sensitive antiviral neutralization bioassay for measuring antibodies to interferons

An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytophathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low OD signals. This fall in OD can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the OD signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte IFN although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques.

A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity

A microtiter cell-culture assay is described for measuring neutralizing antibody activity directed against the human immunodeficiency virus (HIV). The assay relies upon inhibition of HIV-mediated cell killing of infected MT-4 lymphoid cells. The assay exhibits comparable sensitivity to two other methods used for such measurements, is relatively rapid, may be adapted for screening large numbers of samples and involves minimal handling of infectious virus.