Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies

We have identified and characterized nine antigenic epitopes on the E envelope of Japanese encephalitis virus (JEV) by using mAb. Passive administration of most of the anti-JEV mAb protected mice from i.v. challenge with 1.5 x 10(3) plaque-forming units of JEV, JaGAr-01 strain. Some mAb, which possess high neutralization activity in vitro, showed high protection, and JEV-specific N mAb 503 was found the most protective. Even an injection of 2.5 micrograms/mouse of mAb 503 protected all mice from JEV infection. Furthermore, an injection of about 200 micrograms of mAb 503 on day 5 postinfection protected 82% of the mice, even when JEV was detected in more than 85% of the infected mouse brains. Synergism of protection was observed with mixtures of several mAb directed against different epitopes. Although in a murine macrophage cell line, all of the mAb groups showed antibody-dependent enhancement (ADE) of JEV infectivity in vitro, and only two flavivirus cross-reactive mAb groups showed ADE of dengue virus type 2. The ADE of JEV by mAb seems not to be harmful for in vivo protection experiments, except for two mAb groups: mAb 302 and 201 showed little or no protective activity against JEV infection and, rather, caused early death in infected mice.     

Neutralizing Antibodies Protect against Lethal Flavivirus Challenge but Allow for the Development of Active Humoral Immunity to a Nonstructural Virus Protein

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.     

Monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages.

Antibody-dependent enhancement of virus infection is a process whereby virus-antibody complexes initiate infection of cells via Fc receptor-mediated endocytosis. We sought to investigate antibody-dependent enhancement of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro. Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells, by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline antiserum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheep erythrocyte serum and an antibody-coated sheep erythrocyte phagocytosis assay. Thirty-seven murine monoclonal antibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific monoclonal antibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal protein A. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.     

A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.     

Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner

Severe dengue virus infection can occur in humans with pre-existing antibodies against the virus. This observation led to the hypothesis that a subneutralizing antibody level in vivo can increase viral burden and cause more severe disease. Indeed, antibody-dependent enhancement of infection (ADE) in vitro has been described for multiple viruses, including the flaviviruses dengue virus and West Nile virus. Here, we demonstrate that the complement component C1q restricts ADE by anti-flavivirus IgG antibodies in an IgG subclass-specific manner in cell culture and in mice. IgG subclasses that avidly bind C1q induced minimal ADE in the presence of C1q. These findings add a layer of complexity for the analysis of humoral immunity and flavivirus infection.     

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.     

Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.10⁵ or 2.10⁶ TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.     

Antibody-mediated in vitro neutralization of human immunodeficiency virus type 1 abolishes infectivity for chimpanzees.

This study was undertaken to establish whether antibody directed against the human immunodeficiency virus type 1 (HIV-1) principal gp120 type-specific neutralization determinant can abolish the infectivity of HIV-1 in chimpanzees. Challenge inocula of the IIIb virus isolate were mixed in vitro with either immunoglobulin G (IgG) from an uninfected chimpanzee, nonneutralizing IgG from an HIV-seropositive human, a virus-neutralizing murine monoclonal antibody directed against the HIV-1 IIIb isolate, or virus-neutralizing IgG from a chimpanzee infected with the IIIb isolate. Both neutralizing antibodies were directed against the principal neutralization determinant of the challenge isolate. Establishment of infection following inoculation of each virus-antibody mixture into chimpanzees was assessed by virus-specific antibody development and by virus isolation. No protective effect was noted either with the control IgG or with the nonneutralizing anti-HIV IgG. By contrast, the polyclonal chimpanzee virus-neutralizing IgG prevented HIV-1 in vivo infection, while the neutralizing monoclonal antibody notably decreased the infectivity of the challenge virus. Hence, antibody to the gp120 principal neutralization determinant is able both to prevent HIV-1 infection in vitro and to inhibit infection in vivo.     

Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Flavivirus infections are a significant public health problem, since several members of the Flaviviridae family are highly pathogenic to humans. Accurate diagnosis and differentiation of the infecting virus is important, especially in areas where many flaviviruses are circulating. In this study we evaluated a newly developed commercially available immunofluorescence assay (IFA) (INDX, Baltimore, MD, USA) for the detection of IgM and IgG antibodies against dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus. IFA was compared with standard diagnostic enzyme immunoassays (EIAs) specific for the detection of IgM and IgG antibodies against these viruses. Forty-seven serum samples from patients with a defined flavivirus infection were tested. As controls, serum samples from individuals with antibodies against tick-borne encephalitis virus and hepatitis C virus as well as healthy individuals were included. The results obtained from this study indicate that IFA showed a significantly better discrimination for flavivirus specific IgM antibodies than did the standard IgM specific EIAs (the overall cross-reactivity varied between 4 and 10% by IFA and 30-44% by EIA for the respective viruses). In contrast, the detection of flavivirus specific IgG antibodies showed high cross-reactions in both IFA and EIAs (overall cross-reactivity 16-71 and 62-84%, respectively). This study clearly stated the complexity of flavivirus diagnosis, showing that one cannot rely on one assay or search for one virus only. The flavivirus IFA is a useful tool for the identification of flavivirus infections during the acute stage of disease. In particular, IFA can be an important diagnostic tool for testing samples from travellers who have been accidentally exposed to these viruses.     

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m^−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m^−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8⁺ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.     

Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.     

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here, we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses, as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals?a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses.     

Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.     

Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.     

Antiviral peptides targeting the west nile virus envelope protein

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 muM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 muM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.     

Antibodies against West Nile Virus nonstructural protein NS1 prevent lethal infection through Fc gamma receptor-dependent and -independent mechanisms

The flavivirus nonstructural protein NS1 is a highly conserved secreted glycoprotein that does not package with the virion. Immunization with NS1 elicits a protective immune response against yellow fever, dengue, and tick-borne encephalitis flaviviruses through poorly defined mechanisms. In this study, we purified a recombinant, secreted form of West Nile virus (WNV) NS1 glycoprotein from baculovirus-infected insect cells and generated 22 new NS1-specific monoclonal antibodies (MAbs). By performing competitive binding assays and expressing truncated NS1 proteins on the surface of yeast (Saccharomyces cerevisiae) and in bacteria, we mapped 21 of the newly generated MAbs to three NS1 fragments. Prophylaxis of C57BL/6 mice with any of four MAbs (10NS1, 14NS1, 16NS1, and 17NS1) strongly protected against lethal WNV infection (75 to 95% survival, respectively) compared to saline-treated controls (17% survival). In contrast, other anti-NS1 MAbs of the same isotype provided no significant protection. Notably, 14NS1 and 16NS1 also demonstrated marked efficacy as postexposure therapy, even when administered as a single dose 4 days after infection. Virologic analysis showed that 17NS1 protects at an early stage in infection through a C1q-independent and Fc gamma receptor-dependent pathway. Interestingly, 14NS1, which maps to a distinct region on NS1, protected through a C1q- and Fc gamma receptor-independent mechanism. Overall, our data suggest that distinct regions of NS1 can elicit protective humoral immunity against WNV through different mechanisms.     

Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies

In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.     

Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibody-dependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given gamma globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LD50 of SF, a virus which is in a different subgroup than SIN. All mice survived if the gamma globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus. Cobra venom factor treatment of normal C3H mice challenged with SF did not reduce the protection, suggesting that complement was not involved. Methyl palmitate (40 mg/mouse) given before gamma globulin and virus challenge suppressed macrophage activity and reduced the level of protection 23% in females and 70% in males. Silica treatment (3 mg/mouse) reduced the protection equally in both males and females by 92%. In vitro experiments were done to test if it were possible that cross-antibody-dependent cellular cytotoxicity (ADCC) could account for the passive cross-protection observed in this system. Cross-ADCC could be demonstrated in vitro at high dilutions of antiserum (1:25,600). On the basis of the in vitro and in vivo results presented, we suggest that cross-ADCC against SF-infected target cells is one of the likely mechanisms to explain the passive cross-protection observed.     

Epitope determinants of a chimpanzee dengue virus type 4 (DENV-4)-neutralizing antibody and protection against DENV-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody

The chimpanzee monoclonal antibody (MAb) 5H2 is specific for dengue virus type 4 (DENV-4) and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. One variant contained a Lys₁₇₄-Glu substitution and another contained a Pro₁₇₆-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity for the antibody 18- to >100-fold. Humanized immunoglobulin G (IgG) 5H2, originally produced from an expression vector, has been shown to be a variant containing a nine-amino-acid deletion in the Fc region which completely ablates antibody-dependent enhancement of DENV replication in vitro. The variant MAb, termed IgG 5H2 ΔD, is particularly attractive for exploring its protective capacity in vivo. Passive transfer of IgG 5H2 ΔD at 20 μg/mouse afforded 50% protection of suckling mice against challenge with 25 50% lethal doses of mouse neurovirulent DENV-4 strain H241. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge. Monkeys that received 2 mg/kg of body weight of IgG 5H2 ΔD were completely protected against 100 50% monkey infectious doses (MID₅₀) of DENV-4, as indicated by the absence of viremia and seroconversion. A DENV-4 escape mutant that contained a Lys₁₇₄-Glu substitution identical to that found in vitro was isolated from monkeys challenged with 10⁶ MID₅₀ of DENV-4. This substitution was also present in all naturally occurring isolates belonging to DENV-4 genotype III. These studies have important implications for possible antibody-mediated prevention of DENV infection.