Erin Mehlhop

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.

Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus.

WNV continues to spread throughout the Western Hemisphere as virus activity in insects and animals has been reported in the United States, Canada, Mexico, and the Caribbean islands. West Nile virus (WNV) infects the central nervous system and causes severe disease primarily in humans who are immunocompromised or elderly. In this review, we discuss the mechanisms by which the immune system limits dissemination of WNV infection. Recent experimental studies in animals suggest important roles for both the innate and the adaptive immune responses in controlling WNV infection. Interferons, antibody, complement components and CD8+ T cells coordinate protection against severe infection and disease. These findings are analyzed in the context of recent approaches to vaccine development and immunotherapy against WNV.

Protective immune responses against West Nile virus are primed by distinct complement activation pathways

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3−/− and complement receptor 1/2−/− mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

Complement activation is required for induction of a protective antibody response against West Nile virus infection.

ABSTRACT Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.

Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

ABSTRACT Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.

Rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of DC-SIGN

Here, we describe the isolation and characterization of the rhesus macaque homolog for human DC-SIGN, a dendritic cell-specific C-type lectin. mac-DC-SIGN is 92% identical to hu-DC-SIGN. mac-DC-SIGN preserves the virus transmission function of hu-DC-SIGN, capturing and efficiently transducing simian and human immunodeficiency virus to target CD4+ T cells. Surprisingly, however, mac-DC-SIGN plays no discernable role in the ability of rhesus macaque dendritic cells to capture and transmit primate lentiviruses. Expression and neutralization analyses suggest that this process is DC-SIGN independent in macaque, although the participation of other lectin molecules cannot be ruled out. The ability of primate lentiviruses to effectively use human and rhesus dendritic cells in virus transmission without the cells becoming directly infected suggests that these viruses have taken advantage of a conserved dendritic cell mechanism in which DC-SIGN family molecules are significant contributors but not the only participants.

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells.

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4+ T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-α, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4+ T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.

Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses

The macaque-simian immunodeficiency virus (SIV) system is one of the best animal models available to study the role of dendritic cells (DCs) in transmission and pathogenesis of HIV, as well as to test DC-based vaccine and therapeutic strategies. To better define and optimize this system, the responsiveness of macaque monocyte-derived DCs to a variety of maturation stimuli was examined. Characteristic immunophenotypic and functional DC maturation induced by standard monocyte conditioned medium (MCM) was compared to the activation induced by a panel of stimuli including soluble CD40L, LPS, Poly I:C, PGE(2)/TNFalpha, and a cocktail mixture of PGE(2)/TNFalpha/IL-1beta/IL-6. Immunophenotypic analysis confirmed that all stimuli induced stable up-regulation of CD25, CD40, CD80, CD83, CD86, HLA-DR, DC-LAMP (CD208), and DEC-205 (CD205). In general, macaque DCs exhibited weaker responses to LPS and Poly I:C than human DCs, and soluble CD40L stimulation induced variable expression of CD25. Interestingly, while the endocytic capacity of CD40L-matured cells was down-modulated comparably to DCs matured with MCM or the cocktail, the T cell stimulatory activity was not enhanced to the same extent. The particularly reproducible and potent T cell stimulatory capacity of cocktail-treated DCs correlated with a more homogenous mature DC phenotype, consistently high levels of IL-12 production, and better viability upon reculture compared to DCs activated by other stimuli. Furthermore, cocktail-matured DCs efficiently captured and presented inactivated SIV to SIV-primed T cells in vitro. Thus, the cocktail represents a particularly potent and useful stimulus for the generation of efficacious immunostimulatory macaque DCs.

Complement protein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of West Nile virus.

Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remain uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of cells expressing Fc-gamma receptors (CD32) and explains how C1q restricts the ADE of flavivirus infection.