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E J Stott

Publications and source records attributed to E J Stott.

At least 19 recordsLinked to original sources
PubMed1999

Progressive dendritic pathology in cynomolgus macaques infected with simian immunodeficiency virus.

Neuronal pathology in acquired immunodeficiency syndrome (AIDS) is of interest in relation to cognitive impairment in AIDS patients and from the broader perspective of the pathogenesis of neurodegeneration. Cortical dendritic spine loss has been described in patients with AIDS and the aim of this study was to test the hypothesis that similar pathology is present in cynomolgus macaques infected with simian immunodeficiency virus (SIV). These animals develop an AIDS-like illness, but multinucleated giant cell encephalitis is not a feature and CNS virus load is found to be very low. Four animals infected for 2.5-3 months and four infected for 2-3 years were compared with four controls. The Golgi-Cox technique was employed to demonstrate dendritic morphology in the frontal cortex and the diameter of apical dendrites, dendritic spine density and dendritic spine lengths were measured in layer V pyramidal cells. Immunohistochemistry for microtubule-associated protein-2 (MAP-2), MHC class II and glial fibrillary acidic protein (GFAP) was also performed. In infected animals there was progressive spine loss and atrophy of remaining spines with loss of MAP-2 immunoreactivity at late time points. No parallel increase in GFAP immunostaining or MHC-class II expression in microglial cells was seen. We conclude that progressive neuronal dendritic pathology is a feature of SIVmac251 infection of cynomolgus macaques and is apparent relatively early in disease. Furthermore, dendritic abnormalities occur in the absence of either multinucleated giant cell pathology or substantial CNS virus load.

Animals↗
PubMed1998

Changes in neuron size in cynomolgus macaques infected with various immunodeficiency viruses and poliovirus.

Human immunodeficiency virus (HIV) infection leads to clinically significant neuronal pathology, but the underlying mechanism remains unclear. Infection of rhesus macaques with the simian immunodeficiency virus SIVmac251 has been shown to cause atrophy of hippocampal pyramidal cells. The aim of the current investigation was to determine whether SIVmac251 and other viruses with differing abilities to cause immune suppression or encephalitis could cause neuronal atrophy in cynomolgus macaques. Animals infected with SIVmac251 (n = 22), HIV-2 (n = 6). SIVmac239 (n = 7) and poliovirus (n = 10) were investigated, together with 16 controls. Hippocampal pyramidal cell diameter, averaged across the four CA subfields, was reduced by 16.6% in the SIVmac251 group (P < 0.0001) and by 13.3% in the HIV-2 group (P < 0.001), even though the latter virus does not generally cause immunosuppression. Conversely, SIVmac239, which does cause immunosuppression, caused an average neuronal hypertrophy of 6.8% (P = 0.033). Of possible relevance to the different behaviour of the two SIVs is that SIVmac239 is lymphocyte tropic and does not infect CNS microglia in vivo whereas SIVmac251 does. Animals inoculated with poliovirus into the lumbar spinal cord for polio vaccine neurovirulence testing acted as positive controls for CNS inflammation and they also showed an increase in neuronal diameter (4.1%, P = 0.025). The atrophy seen with SIVmac251 and HIV-2 involved all CA subfields but the hypertrophy following SIVmac239 or poliovirus infection was restricted to CA1 and CA2. These observations show a dissociation between the ability of immunodeficiency viruses to cause immune suppression and neuronal pathology and demonstrate that CNS inflammation per se may cause neuronal hypertrophy.

Animals↗
PubMed1998

Evaluation of a candidate human immunodeficiency virus type 1 (HIV-1) vaccine in macaques: effect of vaccination with HIV-1 gp120 on subsequent challenge with heterologous simian immunodeficiency virus-HIV-1 chimeric virus.

Human immunodeficiency virus type 1 (HIV-1) envelope vaccines can now be evaluated for efficacy in macaques by challenging with chimeric viruses in which the env, tat and rev genes of simian immunodeficiency virus (SIV) have been replaced by those of HIV-1. Most experiments have so far been conducted using gp120 molecules derived from T-cell-adapted LAI or MN strains of HIV-1, which predominantly use the CXCR-4 co-receptor. These vaccines protect against infection by apathogenic chimeric virus carrying the same envelope sequences. In the experiment described here, four macaques were vaccinated with W61D gp120 derived from a low passage Dutch isolate and capable of inhibiting the binding of MIP1beta to the co-receptor CCR-5. This vaccine was potent, inducing high titres of binding and neutralizing antibodies against the homologous HIV-1 and tenfold lower titres against a heterologous challenge virus (SHIV(SF33)) in which the env, tat and rev genes of SIV had been replaced by those of a San Francisco isolate, HIV-1(SF33). Despite strong immune responses to the vaccine there was no evidence that it protected against challenge with this chimeric virus. The antigenic divergence between vaccine and challenge virus or the increased virulence of the challenge virus may be responsible for the inability of this vaccine to protect against infection by SHIV(SF33).

AIDS Vaccines↗
PubMed1997

The construction and evaluation of SIV/HIV chimeras that express the envelope of European HIV type 1 isolates.

The molecular construction of SIV/HIV-1 chimeric viruses (or SHIVs), provides a means of infecting macaques with immunodeficiency viruses that express the envelope protein of HIV-1. However, to date, most SHIVs produced express the envelope of isolates of HIV-1 that have been passaged repeatedly in T cell lines. We have taken SHIV-4 and replaced an NheI-AvrII fragment that encompasses the gp120 region and the extracellular portion of gp41 with the equivalent region of two European isolates of HIV-1 (ACH320.3.1 and HIV-1Han-2). Neither of these viruses had been passaged in T cell lines for prolonged periods prior to molecular cloning. Virus stocks were prepared of both SHIV constructs. In vitro, the relative ability of each clone to replicate in four T cell lines mirrored closely the pattern observed with the parental virus donating the envelope sequences. In vivo, only one of the chimeric viruses was infectious in cynomolgus macaques and its recovery was transient. The factors that affect the replication of SHIVs in vitro and in vivo are discussed.

Animals↗
PubMed1997

Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum.

To evaluate its role in protection, immune serum was collected from four macaques which were chronically infected with live attenuated simian immunodeficiency virus (SIVmacC8) and had resisted challenge with wild-type SIVmacJ5. The immune serum was transferred to two naive cynomolgus macaques by intraperitoneal injection (11 ml/kg). Four control macaques received an intraperitoneal injection of normal saline. One day later, all macaques were challenged with 10 MID50 of the J5M challenge stock of SIV. After challenge, all macaques became infected as determined by virus co-culture and diagnostic PCR. Virus loads in PBMC at 2 weeks post-challenge were indistinguishable between the two groups of macaques. Thus, the failure of passive immunization to transfer protection indicates that serum components alone are not sufficient to mediate the potent protection obtained using live attenuated vaccines. This is the first time that serum has been transferred from animals known to be protected against superinfection.

Animals↗
PubMed1996

Strategies for AIDS vaccines.

In the global AIDS epidemic, over half of all infections have occurred in people less than 25 years old resulting in profound social, economic and demographic consequences. Current estimates indicate that the present 15 million HIV infections will increase to over 30 million by the end of the millennium. For most countries a safe and effective vaccine offers the only hope of controlling the spread of this disease. The development of an effective vaccine against HIV is beset with formidable obstacles. Despite these difficulties, substantial progress has been made towards developing effective strategies for vaccination. Human clinical trials and animal models for AIDS, particularly simian immunodeficiency virus (SIV) infection of macaques, have proved invaluable in this quest. Inactivated virus vaccines induced potent protection in this model, but subsequent studies revealed that protection was mediated by antibody to cellular proteins present in the vaccine preparations and on the surface of infecting virions. This surprising observation has provided an alternative and complementary approach to the development of vaccines against HIV in man which is still being pursued. Live attenuated vaccines were initially dismissed as far too hazardous. However, the concept has recently been reexamined in the light of powerful evidence that attenuated SIV induces potent protection against a wide variety of viruses administered by intravenous or mucosal routes and even against challenge with viable virus-infected spleen cells. Efforts are now underway to understand the mechanism of this protection and to attempt to reproduce it by less hazardous means. Considerable effort has been devoted to the development of subunit HIV vaccines, predominantly based on the envelope glycoproteins of the virus. Extensive clinical trials in human volunteers have established that these vaccines are safe and antigenic. However, the immune responses appear to be transient and the antibodies induced do not neutralize the primary isolates of HIV which are circulating in the population. There are now three possible approaches to an AIDS vaccine which are being actively pursued.

AIDS Vaccines↗
PubMed1995

Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells.

A vaccine against AIDS will probably have to protect against challenge both by viable virus-infected cells and by cell-free virus. Eight cynomolgus macaques infected with attenuated simian immunodeficiency virus (SIV) were challenged (four each) with cell-free and cell-associated SIV. All were protected, whereas eight controls were all infected after challenge. These findings show that live-attenuated vaccine can confer protection against SIV in macaques. Extrapolation to human beings will require extensive evaluation of the safety of attenuated retroviruses. Alternatively, the mechanism of this potent protection must be understood and reproduced by less hazardous means.

AIDS Vaccines↗
PubMed1994

Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques.

To characterize the serological response to SIV envelope, induced by vaccination with different envelope immunogens or by SIV infection, plasma samples from 11 cynomolgus macaques infected with simian immunodeficiency virus (SIV) and from 16 macaques vaccinated with three different recombinant envelope proteins were analyzed by (1) ELISA, using a variety of antigens including overlapping peptides encompassing the entire sequence of the envelope protein of SIV, and (2) competition assays, using neutralizing monoclonal antibodies to SIV gp120. Seven regions of SIV envelope were predicted to be antigenic. Peptides representing four of these, in the second and third variable regions (V2 and V3) and the fourth constant (C4) region of gp120 and the Gnann region of gp41, were recognized by the majority of sera from infected and vaccinated animals. Additional antigenic regions were identified in the first and fourth variable domains (V1 and V4) and the carboxy terminus (C5) of gp120 and in three additional regions of gp41. Most infected and vaccinated animals made antibodies that competed with the binding of the three conformational MAbs. Among the vaccinated groups, antibodies induced by vaccination with precursor glycoproteins (gp140 or gp160) recognized several additional gp120 epitopes when compared with antibodies induced by external glycoprotein gp130. Sera from infected animals showed a more restricted gp120 response (17 of 46 peptides recognized) compared to animals vaccinated with precursor glycoproteins (31 peptides recognized). The converse was true for antibodies to gp41. Sera from animals vaccinated with recombinant gp140, produced in insect cells, were the only group that failed to compete with the binding of conformational MAbs. Finally, the development of antibodies to specific epitopes of gp120 and gp41 revealed differences between long-term survivors and nonsurvivors, implying that responses to specific epitopes may be important in conferring resistance to disease progression.

Amino Acid Sequence↗
PubMed1994

Effects of natural sequence variation on recognition by monoclonal antibodies neutralize simian immunodeficiency virus infectivity.

The determinants of immune recognition by five monoclonal antibodies (KK5, KK9, KK17, Senv7.1, and Senv101.1) that neutralize simian immunodeficiency virus infectivity were analyzed. These five neutralizing monoclonal antibodies were generated to native SIVmac251 envelope glycoprotein expressed by a vaccinia virus recombinant vector. All five recognize conformational or discontinuous epitopes and require native antigen for optimal recognition. These monoclonal antibodies also recognize SIVmac239 gp120, but they do not recognize gp120 of two natural variants of SIVmac239, 1-12 and 8-22, which evolved during the course of persistent infection in vivo (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843-1854, 1991). Recombinant viruses which were constructed by exchanging variable regions between SIVmac239 and variant 1-12 were used to define domains important for recognition. Radioimmunoprecipitation analysis demonstrated that sequence changes in variable regions 4 and 5 (V4/V5) were primarily responsible for the loss of recognition of the 1-12 variant. Site-specific mutants were used to define precise changes that eliminate recognition by these neutralizing antibodies. Changing N-409 to D, deletion of KPKE, and deletion of KEQH in V4 each resulted in loss of recognition by all five monoclonal antibodies. SIVs with these natural sequence changes are still replication competent and viable. Changing A-417 to T or A/N-417/418 to TK in V4 or Q-477 to K in V5 did not alter recognition detectably. These results define specific, naturally occurring sequence changes in V4 of SIVmac that result in loss of recognition by one class of SIVmac neutralizing antibodies.

Amino Acid Sequence↗
PubMed1992

Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.

Ten new monoclonal antibodies (MAbs) to SIV envelope were produced and characterized. Using a panel of 28 MAbs, 10 antibody binding sites on SIV envelope protein were identified. Seven sites were located in gp120 and three in gp41. Five sites in gp120 and two in gp41 were defined by overlapping peptides. The remaining two sites on gp120 and one on gp41 were distinguished by competition binding assays but could not be defined by overlapping peptides, suggesting that they were discontinuous or conformational epitopes. Five of the 28 MAbs consistently and reliably neutralized the infectivity of SIVmac251. Two of these bound to a peptide (aa171-190) in the V2 region. The remaining three MAbs bound to a conformational epitope on gp120. These two neutralizing epitopes on SIV are analogous to similar epitopes recently described in HIV-1. In contrast, three MAbs binding to the V3 region of SIV failed to neutralize infectivity, suggesting that this region in SIV may by functionally different from the V3 loop in HIV-1.

Amino Acid Sequence↗
PubMed1992

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.

The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.

Antigens, Viral↗
PubMed1992

Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies.

The regions of the fusion protein of respiratory syncytial virus (RSV) that react with neutralizing, fusion-inhibiting and highly protective bovine and murine monoclonal antibodies (MAbs) were mapped by two methods: (i) competitive binding assays and (ii) production and analysis of antibody-escape mutants. Competitive binding assays with 16 murine and 10 bovine MAbs identified 11 antigenic sites on the fusion (F) protein, many of which overlapped extensively, and indicated that cattle, a natural host for RSV, and mice recognize similar epitopes. Neutralizing MAbs identified four sites, two of which were also fusion-inhibiting and highly protective in mice. The pattern of reactivity of antibody-escape mutants with the MAbs confirmed the mapping of the protective epitopes deduced from competitive binding assays. A comparison of the biological properties of MAbs to the F protein indicated that protection against RSV infection correlated with fusion inhibition rather than neutralization titre or complement-dependent lysis of virus-infected cells.

Animals↗
PubMed1992

Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus.

Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.

Antibodies, Monoclonal↗