Virus load and sequence variation in simian retrovirus type 2 infection

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.     

Endemic Viruses of Squirrel Monkeys (Saimiri spp.)

Nonhuman primates are the experimental animals of choice for the study of many human diseases. As such, it is important to understand that endemic viruses of primates can potentially affect the design, methods, and results of biomedical studies designed to model human disease. Here we review the viruses known to be endemic in squirrel monkeys (Saimiri spp.). The pathogenic potential of these viruses in squirrel monkeys that undergo experimental manipulation remains largely unexplored but may have implications regarding the use of squirrel monkeys in biomedical research.Abbreviations: HTLV1, human T-cell leukemia virus type 1; HVS, herpesvirus saimiri; IPF, idiopathic pulmonary fibrosis; SaHV, Saimiriine herpesvirus; SFV, simian foamy virus; SM-CMV, squirrel monkey cytomegalovirus; SMPyV, squirrel monkey polyomavirus; SMRV, squirrel monkey retrovirusThe similarity between the nonhuman primate and human immune systems is a key advantage in the use of nonhuman primates compared with other mammalian models of human disease.^{13,71,88,94,103,113,125} In addition, the diversity of environmental and infectious disease agents encountered by primates is similar to that of humans, providing nonhuman primates a comparable level of biologic complexity.¹ Old World primates, such as macaques and baboons, and New World primates, including squirrel monkeys and marmosets, are commonly used in biomedical research. Squirrel monkeys (Saimiri spp.) are neotropical primates native to the forests of Central and South America. Of the 7 species of squirrel monkey, 3 (S. oerstedii, S. vanzolinii, and S. ustus) are classified as endangered, vulnerable to extinction in the wild, or near threatened, whereas the remaining 4 (S. boliviensis, S. cassiquiarensis, S. macrodon, and S. sciureus) are not endangered, although the S. cassiquiarensis albigena subspecies is near threatened^52,81 (Figure 1). In South America, where squirrel monkeys are indigenous, breeding colonies of S. sciureus have been maintained at the Pasteur Institute in French Guiana and at the Oswaldo Cruz Foundation in Brazil.^7,12 In the United States, the Squirrel Monkey Breeding and Research Resource, an NIH-sponsored national research resource, maintains breeding colonies for S. boliviensis boliviensis, S. sciureus sciureus, and S. boliviensis peruviensis.Open in a separate windowFigure 1.Taxonomy of Saimiri species with associated IUCN designations.^52,81Squirrel monkeys adapt easily to laboratory housing and can be housed in smaller spaces than can Old World primates.¹ Unlike when working with Old World primates, particularly macaques, no additional personnel protective equipment is necessary when working with squirrel monkeys beyond that recommended for working with other New World primates.⁹² Their small size, combined with the reduced need for personnel protective equipment during handling, make squirrel monkeys attractive species for model development and for studies of viral pathogenesis, which cost approximately 30% to 40% less than comparable studies in macaques.¹ The likelihood of zoonotic transmission of infectious pathogens is considerably less than that associated with macaques and the risk of Macacine herpesvirus 1 (B virus) is nonexistent, given that neotropical primates do not harbor this lethal virus.¹ These factors are increasingly important in the current climate of limited grant funding for biomedical research and emphasis on safety for laboratory personnel. The limited availability of immunologic reagents with specificity for neotropical primates has hindered broader use of squirrel monkeys in biomedical research, compared with that of the more commonly used Old World primates. In addition, the small size of neotropical primates limits the volume of blood that can be collected at any one time. To abrogate these limitations, the NIH Nonhuman Primate Reagent Resource (www.nhpreagents.org) provides an increasing repertoire of agents that have been characterized for immunologic studies of neotropical primates.⁸⁹Squirrel monkeys are used in numerous aspects of biomedical research, including studies of viral persistence, neuroendocrinology, infectious diseases, cancer treatments, vaccine development, gene expression, and reproductive physiology.¹¹⁷ The similarity between the squirrel monkey immune system and that of humans means that, as with macaques, there is a high likelihood that research outcomes will recapitulate what occurs in human diseases.^13,71,87,94 This is particularly true for the study of several notable infectious diseases, including malaria, Creutzfeldt–Jakob disease, and human T-cell leukemia virus type 1 (HTLV1) infection.^19,56,128 For these diseases, squirrel monkeys are the model system of choice for studying pathogenesis, experimental treatments, and strategies for prevention.Squirrel monkeys are recognized as some of the most susceptible nonhuman primate species for the experimental transmission of Creutzfeldt–Jakob disease and other transmissible spongiform encephalopathies that cause chronic wasting disease.^11,72,98,130 The experimental infection of squirrel monkeys with HTLV1 has led to their use in vaccine development and chemotherapy research directed against HTLV1.^44,57,58,82 In addition, squirrel monkeys are an important model for studying the immunology of malaria and for testing vaccines against several Plasmodium species.^19,20,68,114 Furthermore, squirrel monkeys have been used in pharmacologic research to raise HDL levels to prevent atherosclerosis and reduce the risk of coronary heart disease.⁶ As the use of squirrel monkeys increases, especially for infectious disease research, accurate information about the endemic viral infections of squirrel monkeys is needed because of the potential for zoonotic transfer of these viruses to humans (and vice versa) and to understand the potential influence these agents may have on research involving other infectious pathogens diseases and immunosuppressive drugs.     

Quality by design approach for viral clearance by protein a chromatography

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Correlates of preserved CD4(+) T cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4(+) T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4(+) T cell count of 1,076 +/- 589/mm(3) despite chronic infection with a highly replicating virus. No correlation was found between CD4(+) T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4(+) T cell counts were a low percentage of circulating effector T cells (CD28(-)CD95(+) and/or IL-7R/CD127(-)) and a high percentage of CD4(+)CD25(+) T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4(+) T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4(+) T cell counts of <500/mm(3), of which two show severe and persistent CD4(+) T cell depletion (<50/mm(3)). Thus, significant CD4(+) T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4(+) T cell homeostasis in this model of infection. The absence of AIDS in these "CD4(low)" naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4(+) T cell depletion.     

Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix

Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.