Induction of HIV-1 IIIb neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV.

A colinearly synthesized peptide consisting of a H-2d restricted T-helper cell epitope of Semliki Forest virus (SFV) and triple repeats of sequence GPGRAF, derived from the V3 domain of HIV-1 strains, was used to immunize BALB/c (H-2d) mice. Pepscan analysis of sera from peptide-immunized mice revealed that the chimaeric peptide GREKFTIRPHYGKEIGPGRAFGPGRAFGPGRAF contains three distinct antibody-reactive sequences GREKFTIR, PHYGKEI and GPGRAF. The chimaeric peptide evoked HIV-1 IIIb neutralizing antibodies in serum as measured in vitro by reduction of syncytia formation and reduction of p24 production as well. So, the T-helper cell epitope of SFV provided help to a small linear neutralization epitope of HIV-1 strains. Interestingly, the T-helper cell epitope alone might induce antibodies cross-reactive with HIV-1 IIIb specific peptide GPGRAFVTIGK which shows some homology (residues underlined) with the antibody-reactive sequence GREKTIR of SFV.

Critical involvement of Tcf-1 in expansion of thymocytes.

T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4- 8- thymocytes are observed at two distinct stages (CD44+ 25+ and CD44- 25-), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1(-/-) progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1(-/-) mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Furthermore, their peripheral T cells have an activated phenotype (increased CD44 and decreased CD62L expression) and proliferate normally in response to Ag or mitogen, suggesting that these cells may have arisen from the early wave of development during embryogenesis and are either long lived or have subsequently been maintained by peripheral expansion. As Tcf-1 is a critical component in the Wnt/beta-catenin signaling pathway, these data suggest that Wnt-like factors play a role in the expansion of double-negative thymocytes.

Epitope polarity and adjuvants influence the fine specificity of the humoral response against Semliki Forest virus specific peptide vaccines.

The humoral response to synthetic peptide vaccines against Semliki Forest virus (SFV) in H-2d BALB/c mice was investigated with the enzyme linked immunosorbent assay and the pepscan technique. The peptide vaccines consisted of amino acid sequences 240-255 (B) and 137-151 (T) of the E2 membrane protein of SFV colinearly synthesized in either orientation T-B or B-T. Sequence B contains an epitope inducing humoral immunity to lethal SFV infection and sequence T contains a H-2d restricted T-helper cell epitope. With sera from mice immunized subcutaneously with peptide T-B, and Quil A as adjuvant, two immunodominant B-cell epitopes were identified, FVPRAD, at position 240-246 and PHYGKEI, at position 145-151. However, with peptide B-T and Quil A as adjuvant for immunization the epitope PHYGKEI was clearly immunodominant, but antibodies elicited against this epitope were not reactive with SFV-infected L cells in contrast to the antibodies elicited by epitope FVPRAD. An additional epitope EPARKGKVH, at position 247-255, was identified with sera from mice immunized subcutaneously with either peptide T-B or B-T and Montanide ISA 740 as an adjuvant. Monoclonal antibodies selected for reactivity with SFV-infected L cells did bind also to epitope FVPRAD. Interestingly, this epitope could induce antibodies cross-reactive with a synthetic peptide derived from macrophage migration inhibitory factor that shares amino acid residues VPRA at position 9-12 with the protective B-cell epitope FVPRAD. The present study shows clearly that the fine specificity of the humoral response against peptide vaccines is differentially influenced by both adjuvant and epitope polarity which may affect vaccine efficacy. Further, the study reminds us that potentially autoimmune antibodies could be induced by vaccines.

Evaluation of protection by two endotoxin-neutralizing IgM monoclonal antibodies in different peritonitis models.

Two anti-core glycolipid (CGL) IgM monoclonal antibodies (mAbs 8-2 and 26-20), previously shown to display cross-reactivity with heterologous lipopolysaccharide (LPS) in vitro and to provide cross-protectivity against endotoxin challenge in vivo, were evaluated for their potential to protect mice against death from peritonitis caused by heterologous bacterial challenge. Without concurrent antibiotic treatment neither antibody was protective. Compared with a control mAb, prophylactic treatment with mAb 8-2 significantly increased the survival of gentamicin-treated mice challenged with the rough strain Salmonella minnesota Re595. Both mAb 8-2 and a control mAb, in combination with a suboptimal dose of ceftazidime, increased survival following challenge with the clinical isolate Escherichia coli O7:K1. In a model of mucin-enhanced peritonitis, neither mAb was protective against challenge with inocula of E. coli O7:K1, ranging from 10(2) to 10(4) bacteria. We conclude that protection of mice by anti-CGL mAb 8-2 against heterologous challenge is vitally dependent on concurrent treatment with antibiotics and that protection may not be attributable to the anti-CGL specificity of these antibodies.

In vivo TNF induction by culture supernatants of antibiotic-treated Escherichia coli 07:K1. Role of antibiotic class and concentration.

Antibiotics may cause an excess release of lipopolysaccharide (LPS) from bacteria and thereby promote the production of tumour necrosis factor (TNF). TNF was measured in the serum of Swiss mice challenged with filtered supernatant of Escherichia coli O7:K1 that had been exposed to various antibiotics in vitro. Expressed as a function of a standardized number of cells remaining after 6 h of exposure to gentamicin, ceftazidime, ciprofloxacin or imipenem, TNF leves associated with antibiotic exposure always exceeded those of controls. However, if differences in the remaining number of bacteria were not taken into account, TNF induction by supernatant of control untreated cultures was greater than that elicited by supernatant from any of the antibiotic-treated cultures. With the exception of imipenem, low-dose antibiotic exposure (0.5 x MIC) invariably induced higher TNF levels than did high-dose exposure (10 x MIC). Considerable antibiotic class- and concentration-related differences were noted. LAL equivalent amounts of LPS released by different antibiotics may diverge in their capacity to induce TNF. Our results do not support the notion that the use of rapidly bactericidal and lytic antibiotics should be avoided.

A shared idiotope among antibodies against Semliki Forest virus.

In the present study a shared idiotope was found among antibodies against a previously defined linear B-cell epitope of Semliki Forest virus (SFV). The synthetic B-cell epitope, located at amino acid positions 240 to 255 of the E2 membrane protein, was linked to an H-2d-restricted T-helper cell epitope of either SFV or influenza virus. Colinearly synthesized peptides of T-B polarity mixed with adjuvant were used to immunize BALB/c (H-2d) mice. After one booster immunization with either chimaeric peptide high serum antibody titers were measured against both synthetic peptide (240-255) and glutaraldehyde-fixed SFV-infected L cells. Against the synthetic peptide (240-255) a variety of monoclonal antibodies (MAbs) were produced that differed in reactivity with SFV, varied in heavy chain family, isotype, isoelectric point, and idiotype. Against one of the antipeptide MAbs (I02), that strongly reacted with SFV-infected L cells, an antiidiotypic MAb (ab2MAb), designated I02A3, was produced that could be inhibited in its binding to MAb I02 by the synthetic B-cell epitope. Therefore it was concluded that ab2 MAb I02A3 recognizes an idiotope closely associated with the antigen combining site of antipeptide MAb I02. This idiotope was definitively shared by two out of 15 antipeptide MAbs and by SFV-reactive antibodies present in both antipeptide sera and SFV-immune sera.

Influence of epitope polarity and adjuvants on the immunogenicity and efficacy of a synthetic peptide vaccine against Semliki Forest virus.

The antibody response to a previously defined B-cell epitope of Semliki Forest virus (SFV) was investigated in male BALB/c (H-2d) mice. The B-cell epitope, located at amino acid positions 240 to 255 of the E2 protein, was linked to an H-2d-restricted T-helper cell epitope of SFV located at positions 137 to 151 of the E2 protein. Colinearly synthesized peptides, of either T-B or B-T polarity, mixed with different adjuvants (the nonionic block copolymer L 180.5, a water-oil-water [W/O/W] emulsion of L 180.5, Montanide, and Q VAC) were used for immunization. Generally, after one booster immunization, high serum antibody titers were measured against either peptide. With Q VAC and W/O/W L 180.5 as adjuvants, the titers of SFV-reactive (nonneutralizing) antibodies were consistently much higher after immunization with the T-B peptide than with the B-T peptide, which was reflected in a higher vaccine efficacy. With these two adjuvants, the survival ratio in T-B peptide-immunized mice was 82%, compared with 8% in B-T peptide-immunized mice. Intermediate results were obtained with the adjuvant Montanide. L 180.5 alone was ineffective in this study. All immunoglobulin G (IgG) isotypes were induced with either adjuvant, but Q VAC was clearly the most effective in inducing IgG2a and IgG2b isotypes with the T-B peptide as the antigen. Subsequently, monoclonal antibodies (MAbs) of IgM, IgG1, IgG2a, IgG2b, and IgG3 subclasses were prepared against the B-cell epitope. These nonneutralizing but SFV-reactive MAbs protected 40 to 80% of mice against a lethal challenge with SFV. Control mice all died. The availability of those antipeptide MAbs allowed competition binding assays with a previously characterized panel of E2-specific MAbs. Binding of enzyme-labeled antipeptide MAbs was very effectively inhibited by two strongly SFV-neutralizing mutually competitive MAbs, suggesting that the linear B-cell epitope (amino acids 240 to 255) is associated with a major neutralization site of SFV.

Detection of a shared idiotope on two encephalomyocarditis virus neutralizing monoclonal antibodies by neutralization inhibition enzyme immunoassay.

Idiotypic cross-reactivity between encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies UM 21.1 (IgG2b) and UM 21.3 (IgG2a) was detected by neutralization inhibition enzyme immunoassay using polyclonal and monoclonal anti-idiotypic antibodies. One strongly cross-reactive anti-idiotypic monoclonal antibody, designated 21.1A5 (IgG2b), might recognize a recurrent idiotope on EMCV neutralizing antibodies but it did not induce EMCV neutralizing anti-anti-idiotypic antibodies in homologous BALB/c mice.

Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS.

Protection against lethal endotoxemia by anti-lipid A murine monoclonal antibodies: comparison of efficacy with that of human anti-lipid A monoclonal antibody HA-1A.

The protective capacities of murine anti-lipid A monoclonal antibodies (MAbs) 8-2 and 26-20 were examined and compared with those of the human MAb HA-1A with respect to inhibition of lipopolysaccharide (LPS) priming of human polymorphonuclear leukocytes (PMNL) in vitro and protection against lethal endotoxemia in mice. HA-1A did not prevent the priming effect of either rough or smooth LPS, while MAb 26-20 effectively inhibited LPS priming of human PMNL. Also, both murine MAbs protected mice against an otherwise lethal challenge with rough Re LPS of S. minnesota R595 as well as with smooth LPS of E. coli O111:B4. HA-1A exerted no protection against the lethal effects of Re LPS in this in vivo model. The enhanced survival in mice by treatment with MAbs 8-2 and 26-20 was associated with decreased levels of LPS-induced tumor necrosis factor. Neutralization of lipid A as a mechanism of protection was strongly suggested by efficacious inhibition of LPS priming of human PMNL by MAbs 8-2 and 26-20 in vitro.

A delayed-type hypersensitivity-inducing T-cell epitope of Semliki Forest virus mediates effective T-helper activity for antibody production.

The rational development of peptide vaccines requires the identification of both B- and T-cell epitopes. In this study, potential T-helper cell epitopes of Semliki Forest virus (SFV) were identified on the basis of their ability to induce delayed-type hypersensitivity (DTH) in mice using recombinant SFV fragments produced as hybrid proteins with beta-galactosidase in Escherichia coli and synthetic peptides coupled to beta-galactosidase. Although the tested fragments spanned almost the entire amino acid sequence of the structural proteins of SFV, only one DTH-inducing region (located between amino acid 137 and 151 of the SFV E2 membrane protein) was identified. Peptides containing this E2 region stimulated lymph node cells from SFV-primed mice in vitro. The ability of the identified T-cell epitope to induce a specific T-helper response in mice was evaluated using synthetic peptides that contained combinations of the DTH-inducing region and different previously identified linear B-cell epitopes of E2. These peptides proved able to induce an antipeptide IgG response in mice in an H-2d-restricted fashion. One of the peptides was also able to induce high titres of IgG reactive with SFV-infected cells and protected 70-100% of the peptide-immunized mice after challenge with virulent SFV. Our findings suggest that DTH and T-helper activity are mediated by different doses of the same T-cell epitope.

A vaccine against Semliki Forest virus consisting of a monoclonal anti-idiotypic antibody cross-linked to a protein which contains virus-specific T-helper cell epitopes.

A recombinantly expressed protein, consisting of cro-beta-galactosidase at the N-terminus and amino acid residues 115 to 151 of the E2 membrane of Semliki Forest virus (SFV) at the C-terminus containing two T-helper cell epitopes of SFV, was cross-linked with glutaraldehyde to a noninternal image monoclonal anti-idiotypic antibody (ab2 alpha MAb) able to induce SFV-neutralizing anti-anti-idiotypic (ab3) antibodies in BALB/c mice. This vaccine, which might potentially induce SFV-specific T-helper cell memory, established in BALB/c mice a state of protective immunity against virulent SFV within 10 days of immunization. A steady rise in serum neutralization titre occurred from day 7 to day 28 after primary anti-idiotypic immunization, levelling off thereafter. In primarily immunized mice significant rises of serum neutralization titres, which could be indicative for an operational T-helper cell memory, were not observed after challenge on day 35 with virulent SFV. The results suggest that SFV is neutralized by ab3 antibodies shortly after challenge, preventing, thereby, virus multiplication to levels sufficient to provoke a measurable booster response.

Immunogenicity and vaccine efficacy of synthetic peptides containing Semliki Forest virus B and T cell epitopes.

A synthetic peptide that contains a Semliki Forest virus (SFV) B cell epitope, located at amino acid positions 240 to 255 of the E2 protein, and an SFV T helper (Th) cell epitope, located at positions 137 to 151 of the E2 protein, evoked high titres of SFV-reactive antibodies in H-2d mice. Although the peptide-induced antibodies did not neutralize SFV in vitro, 70 to 100% of the peptide-immunized mice were protected against SFV, even when viral challenge was presented 4 months after immunization. The protection could be transferred by anti-peptide serum, indicating that antibodies were responsible for the protection. When the Th cell epitope of this protective peptide was replaced by an influenza virus Th cell epitope or by another SFV Th cell epitope, the resulting peptides induced lower non-neutralizing SFV-reactive antibody titres and protected a correspondingly lower percentage of mice (50% and 30%, respectively). A peptide with the same Th cell epitope as the best protective peptide but with a less effective SFV B cell epitope protected only 33% of the mice. These results indicate that protection against SFV by a synthetic peptide is primarily dependent on its ability to induce adequate amounts of antibodies with relevant specificity and sufficient affinity; the ability to induce a relevant (SFV-specific) T memory response played only a minor role in protection.

Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein.

Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.

Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine.

Semliki Forest virus (SFV) infection of mice was used as a model to study the applicability of synthetic peptides containing only linear epitopes as viral vaccines. The identification of linear epitopes with vaccine potential on the E2 membrane protein of SFV was based on the binding of SFV-specific antibodies to a set of overlapping synthetic hexapeptides (Pepscan) representing the whole E2 amino acid sequence. The 14 available E2-specific monoclonal antibodies which were protective in vivo proved to be unsuitable for the identification of linear epitopes because they recognized only conformational epitopes, as indicated by their lack of reactivity with unfolded, reduced E2 protein on immunoblots. Three epitopes were detected with polyclonal anti-SFV serum at amino acid positions 135 to 141, 177 to 185 and 240 to 246 of the E2 protein. Synthetic peptides containing these epitopes were coupled to a carrier protein and tested as a vaccine. Mice immunized with the peptide containing amino acids 240 to 255 of protein E2 were protected against a challenge with virulent SFV but protection of mice immunized with the peptides containing amino acids 126 to 141 or 178 to 186 was only marginally better than that of controls. The prechallenge sera of most peptide-immunized mice reacted with SFV-infected cells but none of these sera neutralized the virus in vitro. However, protection of mice correlated well with SFV-specific antibody titre, suggesting antibody-mediated protection.

Identification of a DTH-inducing T-cell epitope on the E2 membrane protein of Semliki Forest virus.

Mapping of T-cell epitopes on the structural proteins of Semliki Forest virus (SFV) was performed by measuring the ability of cloned SFV protein fragments to induce delayed-type hypersensitivity (DTH). The cloned SFV protein fragments were expressed as hybrid proteins with cro-beta-galactosidase in Escherichia coli from constructed recombinant plasmids. DTH reactions were measured, as footpad swelling, in BALB/c mice after immunization with whole, UV-inactivated SFV and challenge with the hybrid proteins, and vice versa, using the adjuvant dimethyl dioctadecyl ammonium bromide to enhance DTH. Only two of the tested hybrid proteins induced DTH, and these DTH reactions were equally strong. The largest DTH-inducing hybrid protein contained the N-terminal 350 amino acids of E2 and part of E3, the smallest contained only the region from amino acid residues 115 to 151 of the E2 membrane protein without any other SFV protein parts. It was concluded that the segment between amino acid residues 115 and 151 of the E2 membrane protein of SFV was responsible for the observed DTH, and thus, contains a T-cell epitope. Sequence homology with known T-cell epitopes on other proteins makes it likely that the DTH-inducing T-cell epitope is located from amino acid residues 120 to 128 of E2.

Discrimination of determinant specificity of two encephalomyocarditis virus neutralizing monoclonal antibodies by competition, mixed neutralization and anti-idiotypic antibodies.

To investigate the epitope(s) on encephalomyocarditis virus (EMCV) involved in neutralization, two neutralizing monoclonal antibodies (MAs) (MA UM 21.1 and MA UM 21.2) were tested in a competition binding assay (CBA), a mixed neutralization test and an enzyme immunoassay (EIA) with specificity for the detection of idiotypes on MAs. With a CBA in cell culture, using EMCV infected L cell monolayers as binding antigen, strong homologous competition was observed between unlabelled MAs and horse radish peroxidase (HRPO-) labelled MAs but considerable heterologous competition did also occur, especially between the unlabelled MA UM 21.1 and HRPO-labelled MA UM 21.2. In the mixed neutralization test (50% plaque reduction) preincubation with slightly neutralizing or nonneutralizing doses of MA UM 21.2 had no diminishing effect on the neutralizing capacity of MA UM 21.1, the PRT50 value remains in all cases -10log titre of 5.8. Furthermore rabbit polyclonal antibodies against the idiotypes of MAs UM 21.1 and UM 21.2 did not cross react in the EIA. In conclusion both MAs recognize different viral determinants as indicated by the results obtained with a CBA, a mixed neutralization test and an EIA for detection of idiotypes on MAs.

Prophylaxis and therapy of virulent encephalomyocarditis virus infection in mice by monoclonal antibodies. Brief report.

Two encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies (MAs), recognizing different determinants on EMCV, were both able to protect mice prophylactically and therapeutically against a lethal dose of EMCV.

Discrimination of the determinant specificity of two competitive Semliki Forest virus neutralizing monoclonal antibodies by anti-idiotypic antibodies.

The two highly competitive, monoclonal antibodies (MAs), UM 5.1 and UM 8.1, recognize on the E2 glycoprotein of Semliki Forest virus different determinants as indicated by the absence of cross-reactive anti-idiotypic antibodies in rabbit immune sera induced against these highly neutralizing MAs.

The role of complement in monoclonal antibody-mediated protection against virulent Semliki Forest virus.

Monoclonal antibodies (MAs), specific for either the E1 or E2 glycoproteins of Semliki Forest virus (SFV), and belonging to various immunoglobulin subclasses (IgM, IgG2a, IgG2b and IgG3), effected lysis of SFV-infected L cells in co-operation with guinea-pig complement. In this antibody-dependent complement-mediated cytolysis (ADCMC) test, IgG1 MAs were not effective although these antibodies recognize the viral antigens on the surface of SFV-infected L cells. The latter was shown with horseradish peroxidase (HRPO)-labelled MAs in a direct enzyme immunoassay. The binding reactivities of HRPO-labelled MAs to infected L cells at selected time-intervals after infection correlated well with the amount of cytolysis in a parallel ADCMC test. Cytolysis was dependent on the duration of incubation with antibodies: more cytolysis was measured after a 4-hr incubation period with MA, starting at 4 hr after infection, compared to a 1-hr incubation period starting after 7 hr of infection. However, in the latter case (1-hr period) the amount of cytolysis measured correlated better to neutralization and/or protection by MAs than after the extended period (4 hr) of incubation. Complement (C3) depletion by cobra venom factor treatment led to a higher mortality and viraemia of mice prophylactically injected with critically protective doses of either the neutralizing MA UM 8.4 (IgM) or the non-neutralizing MA UM 4.2 (IgG2a). The results suggest a co-operative role of MA with complement in mediating protection against SFV. Passive immunization by administration of low amounts (0.1 micrograms/mouse) of neutralizing MA UM 5.1 resulted in protection of normal mice against a lethal infection with SFV. Mice immunosuppressed by cyclophosphamide were not protected by these doses. If the doses were increased however, these mice were protected both prophylactically and therapeutically. These results indicate that, using critical doses of MAs, an intact immune system ensures survival in normal mice after infection with virulent SFV.