Robert E. Means

Viral Infection of the Placenta Leads to Fetal Inflammation and Sensitization to Bacterial Products Predisposing to Preterm Labor

Pandemics pose a more significant threat to pregnant women than to the nonpregnant population and may have a detrimental effect on the well being of the fetus. We have developed an animal model to evaluate the consequences of a viral infection characterized by lack of fetal transmission. The experiments described in this work show that viral infection of the placenta can elicit a fetal inflammatory response that, in turn, can cause organ damage and potentially downstream developmental deficiencies. Furthermore, we demonstrate that viral infection of the placenta may sensitize the pregnant mother to bacterial products and promote preterm labor. It is critical to take into consideration the fact that during pregnancy it is not only the maternal immune system responding, but also the fetal/placental unit. Our results further support the immunological role of the placenta and the fetus affecting the global response of the mother to microbial infections. This is relevant for making decisions associated with treatment and prevention during pandemics.

Vaccine Protection against Simian Immunodeficiency Virus by Recombinant Strains of Herpes Simplex Virus

ABSTRACT An effective vaccine for AIDS may require development of novel vectors capable of eliciting long-lasting immune responses. Here we report the development and use of replication-competent and replication-defective strains of recombinant herpes simplex virus (HSV) that express envelope and Nef antigens of simian immunodeficiency virus (SIV). The HSV recombinants induced antienvelope antibody responses that persisted at relatively stable levels for months after the last administration. Two of seven rhesus monkeys vaccinated with recombinant HSV were solidly protected, and another showed a sustained reduction in viral load following rectal challenge with pathogenic SIVmac239 at 22 weeks following the last vaccine administration. HSV vectors thus show great promise for being able to elicit persistent immune responses and to provide durable protection against AIDS.

Ability of the V3 Loop of Simian Immunodeficiency Virus To Serve as a Target for Antibody-Mediated Neutralization: Correlation of Neutralization Sensitivity, Growth in Macrophages, and Decreased Dependence on CD4

ABSTRACT To better define the effects of sequence variation and tropism on the ability of the simian immunodeficiency virus SIVmac V3 loop to act as a target of antibody-mediated neutralization, a series of experiments were performed. Three SIV strains, SIVmac239, SIVmac316, and SIVmac155/T3, each with defined differences in envsequence and tropism, were used to construct a panel of viruses chimeric for a portion of envelope that includes the V2 and V3 regions. Peptides with sequences corresponding to the V3 loops of the parental viruses were used to immunize rabbits. The polyclonal rabbit antibodies and plasma from SIVmac239-infected animals were then used to assess the neutralization sensitivity of the parental and chimeric viruses. One of the parental viruses, SIVmac316, which is able to replicate to high titer in alveolar macrophages and can infect cells in a CD4-independent fashion, was highly sensitive to neutralization by plasma from SIVmac-infected rhesus macaques, with average 50% neutralization titers of 1:20,480; this same strain was also sensitive to neutralization by the anti-V3 loop peptide sera. Other parental and chimeric viruses were less sensitive to neutralization with this same panel of antibodies, but as seen with SIVmac316, those viruses that were able to productively replicate in alveolar macrophages were more sensitive to antibody-mediated neutralization. To further define the amino acids involved in increased sensitivity to neutralization, a panel of viruses was constructed by changing envelope residues in SIVmac316 to the corresponding SIVmac239 amino acids. The increased neutralization sensitivity observed for SIVmac316 was mapped principally to three amino acid changes spread throughout gp120. In addition, the increased sensitivity to neutralization by V3-directed antibodies correlated with the ability of the various viruses to replicate to high levels in alveolar macrophage cultures and a CD4-negative cell line, BC7/CCR5. These results demonstrate that the V3 loop of SIVmac Env can act as an efficient target of neutralizing antibodies in a fashion that is highly dependent on sequence context. In addition, these studies suggest a correlation between decreased dependence on CD4 and increased sensitivity to antibody-mediated neutralization.

Downregulation of Gamma Interferon Receptor 1 by Kaposi's Sarcoma-Associated Herpesvirus K3 and K5

ABSTRACT Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway. In order to complete their life cycles, viruses must modulate the host IFN-mediated immune response. The K3 and K5 proteins of a human tumor-inducing herpesvirus, Kaposis sarcoma-associated herpesvirus (KSHV), have been shown to downregulate the surface expression of host immune modulatory receptors by increasing their endocytosis rates, which leads to suppression of cell-mediated immunity. In this report, we demonstrate that K3 and K5 both specifically target gamma interferon receptor 1 (IFN-γR1) and induce its ubiquitination, endocytosis, and degradation, resulting in downregulation of IFN-γR1 surface expression and, thereby, inhibition of IFN-γ action. Mutational analysis indicated that K5 appeared to downregulate IFN-γR1 more strongly than K3 and that the amino-terminal ring finger motif and the carboxyl-terminal region of K5 were necessary for IFN-γR1 downregulation. These results suggest that KSHV K3 and K5 suppress both cytokine-mediated and cell-mediated immunity, which ensures efficient viral avoidance of host immune controls.

Kaposi's Sarcoma-Associated Herpesvirus OX2 Glycoprotein Activates Myeloid-Lineage Cells To Induce Inflammatory Cytokine Production

ABSTRACT Kaposis sarcoma is an inflammatory cytokine-mediated angioproliferative disease which is triggered by infection by Kaposis sarcoma-associated herpesvirus (KSHV). KSHV contains an open reading frame, K14, that has significant homology with cellular OX2, designated viral OX2 (vOX2). In this report, we demonstrate that vOX2 encodes a glycosylated cell surface protein with an apparent molecular mass of 55 kDa. Purified glycosylated vOX2 protein dramatically stimulated primary monocytes, macrophages, and dendritic cells to produce the inflammatory cytokines interleukin 1β (IL-1β), IL-6, monocyte chemoattractant protein 1, and TNF-α. Furthermore, expression of vOX2 on B lymphocytes stimulated monocytes to produce inflammatory cytokines in mixed culture. These results demonstrate that like its cellular counterpart, vOX2 targets myeloid-lineage cells, but unlike cellular OX2, which delivers a restrictive signal, KSHV vOX2 provides an activating signal, resulting in the production of inflammatory cytokines. Thus, this is a novel viral strategy where KSHV has acquired the cellular OX2 gene to induce inflammatory cytokine production, which potentially promotes the cytokine-mediated angiogenic proliferation of KSHV-infected cells.

Viral Infection of the Pregnant Cervix Predisposes to Ascending Bacterial Infection

Preterm birth is the major cause of neonatal mortality and morbidity, and bacterial infections that ascend from the lower female reproductive tract are the most common route of uterine infection leading to preterm birth. The uterus and growing fetus are protected from ascending infection by the cervix, which controls and limits microbial access by the production of mucus, cytokines, and antimicrobial peptides. If this barrier is compromised, bacteria may enter the uterine cavity, leading to preterm birth. Using a mouse model, we demonstrate, to our knowledge for the first time, that viral infection of the cervix during pregnancy reduces the capacity of the female reproductive tract to prevent bacterial infection of the uterus. This is due to differences in susceptibility of the cervix to infection by virus during pregnancy and the associated changes in TLR and antimicrobial peptide expression and function. We suggest that preterm labor is a polymicrobial disease, which requires a multifactorial approach for its prevention and treatment.

Placental Viral Infection Sensitizes to Endotoxin‐Induced Pre‐Term Labor: A Double Hit Hypothesis

Citation Cardenas I, Mor G, Aldo P, Lang SM, Stabach P, Sharp A, Romero R, Mazaki‐Tovi S, Gervasi MTeresa, Means RE. Placental viral infection sensitizes to endotoxin‐induced pre‐term labor: a double hit hypothesis. Am J Reprod Immunol 2011; 65: 110–117

Multiple endocytic trafficking pathways of MHC class I molecules induced by a Herpesvirus protein

The K3 protein of a human tumor‐inducing herpesvirus, Kaposis sarcoma‐associated herpesvirus (KSHV), down‐regulates major histocompatibility complex (MHC) class I surface expression by increasing the rate of endocytosis. In this report, we demonstrate that the internalization of MHC class I by the K3 protein is the result of multiple, consecutive trafficking pathways that accelerate the endocytosis of class I molecules, redirect them to the trans‐Golgi network (TGN), and target MHC class I to the lysosomal compartment. Remarkably, these actions of K3 are functionally and genetically separable; the N‐terminal zinc finger motif and the central sorting motif are involved in triggering internalization of MHC class I molecules and redirecting them to the TGN. Subsequently, the C‐terminal diacidic cluster region of K3 is engaged in targeting MHC class I molecules to the lysosomal compartment. These results demonstrate a novel trafficking mechanism of MHC class I molecules induced by KSHV K3, which ensures viral escape from host immune effector recognition.

Determinants of Increased Replicative Capacity of Serially Passaged Simian Immunodeficiency Virus with nef Deleted in Rhesus Monkeys

ABSTRACT Most rhesus macaques infected with simian immunodeficiency virus SIVmac239 with nef deleted (either Δnef or ΔnefΔvprΔUS [Δ3]) control viral replication and do not progress to AIDS. Some monkeys, however, develop moderate viral load set points and progress to AIDS. When simian immunodeficiency viruses (SIVs) recovered from two such animals (one Δnef and the other Δ3) were serially passaged in rhesus monkeys, the SIVs derived from both lineages were found to consistently induce moderate viral loads and disease progression. Analysis of viral sequences in the serially passaged derivatives revealed interesting changes in three regions: (i) an unusually high number of predicted amino acid changes (12 to 14) in the cytoplasmic domain of gp41, most of which were in regions that are usually conserved; these changes were observed in both lineages; (ii) an extreme shortening of nef sequences in the region of overlap with U3; these changes were observed in both lineages; and (iii) duplication of the NF-κB binding site in one lineage only. Neither the polymorphic gp41 changes alone nor the U3 deletion alone appeared to be responsible for increased replicative capacity because recombinant SIVmac239Δnef, engineered to contain either of these changes, induced moderate viral loads in only one of six monkeys. However, five of six monkeys infected with recombinant SIVmac239Δnef containing both TM and U3 changes did develop persisting moderate viral loads. These genetic changes did not increase lymphoid cell-activating properties in the monkey interleukin-2-dependent T-cell line 221, but the gp41 changes did increase the fusogenic activity of the SIV envelope two- to threefold. These results delineate sequence changes in SIV that can compensate for the loss of the nef gene to partially restore replicative and pathogenic potential in rhesus monkeys.

Herpesviral protein targets a cellular WD repeat endosomal protein to downregulate T lymphocyte receptor expression.

Herpesvirus saimiri Tip associates with Lck and downregulates Lck signal transduction. Here we demonstrate that Tip targets a lysosomal protein p80, which consists of an N-terminal WD repeat domain and a C-terminal coiled-coil domain. Interaction of Tip with p80 facilitated lysosomal vesicle formation and subsequent recruitment of Lck into the lysosomes for degradation. Consequently, Tip interactions with Lck and p80 result in downregulation of T cell receptor (TCR) and CD4 surface expression. Remarkably, these actions of Tip are functionally and genetically separable: the N-terminal p80 interaction is responsible for TCR downregulation and the C-terminal Lck interaction is responsible for CD4 downregulation. Thus, lymphotropic herpesvirus has evolved an elaborate mechanism to deregulate lymphocyte receptor expression to disarm host immune control.