Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques.

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.

Multifidus muscle cross-sectional area adaptations over two volleyball seasons and one off-season in athletes with and without low back pain.

BACKGROUND: The multifidus muscle is important in spine stabilization. Atrophy of the multifidus muscle has been associated with low back pain. OBJECTIVE: To examine multifidus muscle cross-sectional area (CSA) adaptations over two volleyball seasons and one off-season in volleyball athletes experiencing low back pain or no low back pain. METHODS: Twelve female NCAA division 1 volleyball athletes participated. Athletes were placed into a low back pain or no low back pain group. Athlete's multifidus was imaged and measured using ultrasound at four time points across seasons. Imaging time points were before season one, following season one, following off-season, and following season two. A single level mixed-model analysis of variance was used for all analyses. A Tukey HSD post hoc test was used to determine differences between and within the low back pain and the no low back pain groups. RESULTS: Following off-season training the pain group had clinically significant smaller multifidus CSA at the L4 (-2.36 cm2 difference or 17.5%) and L5 (-2.40 cm2 or 12.5%) levels. Non-significant (p> 0.05) decreases in multifidus CSA were seen in both groups following season one and two. Athletes with pain had decreased multifidus CSA at the L4 and L5 vertebral levels at all time points which was non-significant (p> 0.05). CONCLUSIONS: Clinically significant decreases in multifidus CSA occurred in female volleyball athletes with low back pain at the L4 and L5 level following off-season training. Volleyball athletes with pain had smaller multifidus CSA averages at all time points measured of the two year period.

A comparison of neonatal Gram-negative rod and Gram-positive cocci meningitis.

OBJECTIVE: Neonatal meningitis is an illness with potentially devastating consequences. Early identification of potential risk factors for Gram-negative rod (GNR) infections versus Gram-positive cocci (GPC) infection prior to obtaining final culture results is of value in order to appropriately guide expirical therapy. We sought to compare laboratory and clinical parameters of GNR and GPC meningitis in a cohort of term and premature infants. STUDY DESIGN: We evaluated lumbar punctures from neonates cared for at 150 neonatal intensive care units managed by the Pediatrix Medical Group Inc. We compared cerebrospinal fluid (CSF) parameters (white blood cell count, red blood cell count, glucose, and protein), demographics, and outcomes between infants with GNR and GPC meningitis. CSF cultures positive with coagulase-negative staphylococci were excluded. RESULTS: We identified 77 infants with GNR and 86 with GPC meningitis. There were no differences in gestational age, birth weight, infant sex, race, or rate of Caesarean section. GNR meningitis was more often diagnosed after the third postnatal day and was associated with higher white blood cell and red blood cell counts. GNR meningitis diagnosed in the first 3 days of life was associated with antepartum antibiotic exposure. No difference was noted in either CSF protein or glucose levels. After correcting for gestational age, there was no observed difference in mortality between infants infected with GNR or GPC. CONCLUSION: Compared to GPC meningitis, GNR meningitis was associated with several aspects of the clinical history and laboratory findings including older age of presentation, antepartum exposure to antibiotics, and elevated CSF white blood cell and red blood cell counts.

The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

HIV type 1 V3 region primer-induced antibody suppression is overcome by administration of C4-V3 peptides as a polyvalent immunogen.

The extreme variability of HIV-1 immunogenic regions has hampered attempts to design immunogens capable of inducing broadly reactive neutralizing anti-HIV antibody responses. We have begun to study the immune responses generated to a polyvalent mixture of HIV envelope gp120 synthetic peptides, and to determine the ability of each component of a polyvalent immunogen to prime and boost immune responses to each immunogen component. A major concern regarding the use of a polyvalent mixture of HIV-1 immunogens is that the phenomenon of "original antigenic sin," or HIV-1 primer-induced suppression of antibody responses to a subsequent boost by a second HIV-1 variant, may occur and prevent effective anti-HIV immune responses. Using a prototypic four-valent HIV peptide envelope immunogen in BALB/c mice, we observed two types of primer-induced antibody suppression: "original antigenic sin" with primer-induced suppression of antibody responses to only the boosting immunogen, and a second, novel form of primer-induced antibody suppression, with inhibition of antibody responses not only to the priming immunogen but also to all other immunogens in the polyvalent immunogen mixture as well. Importantly, either reversing the sequence of administration of the immunogens or administration of all four components as a polyvalent mixture completely overcame both forms of HIV-1 primer-induced antibody suppression.

HIV-1 gp41 envelope IgA is frequently elicited after transmission but has an initial short response half-life.

Prevention of HIV-1 transmission at mucosal surfaces will likely require durable pre-existing mucosal anti-HIV-1 antibodies (Abs). Defining the ontogeny, specificities and potentially protective nature of the initial mucosal virus-specific B-cell response will be critical for understanding how to induce protective Ab responses by vaccination. Genital fluids from patients within the earliest stages of acute HIV-1 infection (Fiebig I-VI) were examined for multiple anti-HIV specificities. Gp41 (but not gp120) Env immunoglobulin (Ig)A Abs were frequently elicited in both plasma and mucosal fluids within the first weeks of transmission. However, shortly after induction, these initial mucosal gp41 Env IgA Abs rapidly declined with a t(½) of ∼2.7 days. B-cell-activating factor belonging to the TNF family (BAFF) was elevated immediately preceding the appearance of gp41 Abs, likely contributing to an initial T-independent Ab response. HIV-1 transmission frequently elicits mucosal HIV-1 envelope-specific IgA responses targeted to gp41 that have a short half-life.

NMR-derived solution conformations of a hybrid synthetic peptide containing multiple epitopes of envelope protein gp120 from the RF strain of human immunodeficiency virus.

Solution conformations of a 40-residue hybrid peptide containing T-helper epitopes and B-cell determinants from envelope glycoprotein gp120 of human immunodeficiency virus (HIV) have been investigated with NMR. Peptides of this general design are highly immunogenic and induce HIV-neutralizing antibodies and T-lymphocyte responses. The 16-residue N-terminal segment of the peptide contains a T-helper epitope, while the 24-residue C-terminal segment is derived from the V3 loop of HIV strain RF and contains epitopes that elicit neutralizing antibodies as well as T-cell responses. On the basis of 2D proton NMR spectra (COSY, TOCSY, and NOESY) of the peptide in aqueous solution, the resonances of nearly all hydrogens are assigned. The peptide is largely disordered, but specific medium-range NOEs demonstrate conformational preferences in certain regions. Part of the N-terminal segment exhibits nascent helical conformation, consistent with a finding that many T-cell antigens can be modeled as amphipathic helices. In the V3-derived segment of the peptide, one region shows evidence of a tight turn conformation, corresponding to a turn found previously in V3 peptides of HIV strains MN and IIIB. Other conformational features are also detected in the V3 region, such as a stretch of beta strand and a kink that may arise from side-chain interactions.

Conformational preferences of a chimeric peptide HIV-1 immunogen from the C4-V3 domains of gp120 envelope protein of HIV-1 CAN0A based on solution NMR: comparison to a related immunogenic peptide from HIV-1 RF.

A critical problem to overcome on HIV vaccine design is the variability among HIV strains. One strategy to solve this problem is the construction of multicomponent immunogens reflective of common HIV motifs. Currently, it is not known if these motifs should be based primarily on amino acid sequence or higher-order structure of the viral proteins of a combination of the two. In this paper, we report NMR-derived solution conformations for a sympathetic peptide taken from the C4 and V3 domains of HIV-1 CAN0A gp120 envelope protein. This peptide, designated T1-SP10CAN0(A), is compared to a recently reported C4-V3 peptide. T1-SP10RF(A) from the HIV-1 RF strain [de Lorimier et al. (1994) Biochemistry 33, 2055-2062], in terms of conformational features and immune responses in mice [Haynes et al. (1995) AIDS Res. Hum. Retroviruses 11, 211-221]. The T1 segment of 16 amino acids from the gp120 C4 domain is identical in both peptides and exhibits nascent helical character. The SP10 region, taken from the gp120 V3 loop, differs from that of T1-SP10RF(A) in both sequence and conformations. A reverse turn is observed at the conserved GPGX sequence. The rest of the Sp10 domain is extended with the exception of the last three residues which show evidence for a helical arrangement. Modeling of the turn region of the T1-SP10CAN0(A) peptide shows exposure of a continuous apolar stretch of side chains similar to that reported in the crystal structure of a V3 peptide from HIV-1 MN complexed with a monoclonal antibody [Rini et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6325-6329]. this hydrophobic patch is interrupted by a charged Lys residue in the T1-SP10RF(A) peptide. This observation suggests that the HIV-1 CAN0A and HIV-1 RF C4-V3 peptides can induce widely different anti-HIV antibodies. consistent with immunogenic results.

The PIG-A mutation and absence of glycosylphosphatidylinositol-linked proteins do not confer resistance to apoptosis in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder characterized by complement-mediated hemolysis and deficient hematopoiesis. The development of PNH involves an acquired mutation in the X-linked PIG-A gene, which leads to incomplete bioassembly of glycosylphosphatidylinositol (GPI) anchors and absent or reduced surface expression of GPI-linked proteins. The origin and mechanisms by which the PNH clone becomes dominant are not well understood, but recently resistance to apoptosis has been postulated. To test the hypothesis that the PIG-A mutation and absence of GPI-linked surface proteins directly confer resistance to apoptosis, we isolated peripheral granulocytes from 26 patients with PNH and 20 normal controls and measured apoptosis induced by serum starvation. Granulocytes from patients with PNH were relatively resistant to apoptosis (38.8% +/- 14.1%) as compared with granulocytes from controls (55.0% +/- 12.0%, P < .001). However, this resistance to apoptosis was not related to the dominance of the PNH clone because patients with a low percentage of GPI-deficient granulocytes had a similar rate of apoptosis as those with a high percentage of GPI-deficient granulocytes. Similarly, the resistance to granulocyte apoptosis was not influenced by the degree of neutropenia or a prior history of aplastic anemia. To investigate formally the importance of GPI-linked surface proteins in apoptosis, we introduced the PIG-A cDNA sequence into the JY5 GPI-negative B-lymphoblastoid cell line using two different methods: (1) stable transfection of a plasmid containing PIG-A, and (2) stable transduction of a retroviral vector containing PIG-A. We then measured rates of apoptosis induced either by Fas antibody, serum starvation, or gamma-irradiation. With each stimulus, apoptosis of JY5 with stable surface expression of GPI-linked proteins was not statistically different from the parent JY5 cell line or the JY25 (GPI-positive) cell line. Our data confirm that granulocytes from patients with PNH have a relative resistance to apoptosis as compared with normal granulocytes. However, this resistance does not vary with the level of expression of GPI-linked proteins, and stable introduction of PIG-A cDNA with correction of GPI-linked surface expression does not change the rate of apoptosis. Taken together, our data do not support the hypothesis that the PIG-A mutation and absence of GPI-linked surface proteins directly confer resistance to apoptosis in PNH. We conclude that the resistance to apoptosis in PNH is not related to the PIG-A mutation, indicating that other factors must be important in the origin of this phenomenon and the clonal dominance observed in PNH.

Double beta-lactam regimen compared to an aminoglycoside/beta-lactam regimen as empiric antibiotic therapy for febrile granulocytopenic cancer patients.

In a prospective, randomized trial, 205 febrile episodes in granulocytopenic cancer patients were treated with ceftazidime with or without tobramycin (C +/- T), both agents being administered only if the initial granulocyte count was below 200/microliters, or ceftazidime plus piperacillin (C + P). The overall response rate was 71% (39 of 60 for C +/- T and 45 of 58 for C + P). Logistic regression analyses documented no evidence of a significant difference between the two regimens in overall treatment effect after accounting for the linear effects of potentially important variables, such as infection type and granulocyte count. Although the response rates for the subgroup of patients with bacteremias was better with the C + P regimen (P = 0.06), there was no difference in response for patients with bacteremia and profound (< 100/microliters) sustained granulocytopenia. The double beta-lactam combination demonstrated in vitro synergism in 73%; antagonism was not seen. Both regimens produced excellent serum bactericidal levels (C +/- T geometric mean peak 1:170; C + P peak 1:137) against gram-negative but not gram-positive pathogens (1:4; 1:7 respectively) that had caused bacteremia. Emergence of resistance and significant coagulopathy and/or bleeding did not occur during therapy. Antibiotic-related nephrotoxicity was noted in 7 of 95 trials in the C + P and in 6 of 89 trials in the C +/- T group (P = 0.19). The incidence of secondary infections in patients with profound (< 100/microliters) sustained granulocytopenia was lower in the C +/- T group (P = 0.04). Alimentary canal anaerobic flora preservation with C +/- T, and suppression with C + P, was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy.

It has been well documented that the gamma-diketone HD1 is the ultimate toxic metabolite of n-hexane. Furthermore, it has been shown that the pathogenetic mechanism by which HD exerts its neurotoxic effects is through binding to protein lysly residues and cyclization to pyrroles. The present study sought to determine whether the presence of pyrrole residues on NF1 proteins is sufficient to cause the NF-filled axonal swellings associated with n-hexane and other gamma-diketone neuropathies or whether pyrrole oxidation and protein cross-linking also have to occur in order for neurotoxicity to develop. We synthesized the HD analogue AcHD1 and assessed its rate of pyrrole formation in vitro, the ease of oxidation of its resulting pyrroles, and its ability to cross-link proteins in vitro. The in vivo effects of AcHD on rats were examined following daily ip1 injections. AcHD was found to have a rate of pyrrole formation comparable to that of the potent HD analogue DMHD1 at 35 degrees C. The pyrrole derived from AcHD was more resistant to oxidation than that derived from the neurotoxic compound HD. AcHD did not cross-link proteins in vitro. Pyrrole derivatives were demonstrated on hemoglobin isolated from animals treated with HD, DMHD, and AcHD. Cross-linked spectrin was detected in animals treated with HD and DMHD but not with AcHD. Rats receiving 0.1 or 0.25 mmol of AcHD/kg/day did not reach the end point of hindlimb paralysis observed in the gamma-diketone neuropathies, and the NF-filled axonal swellings seen following exposure to the neurotoxic gamma-diketones were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus sinusitis in cancer patients.

Paranasal sinusitis occurred in 52 immunosuppressed cancer patients treated over 5 years at the University of Maryland Cancer Center. Twenty-one patients had aspergillus sinusitis; Aspergillus sp, including flavus and niger were directly recovered from sinus in 19 of the 21 infections. Two other patients with sinus involvement and positive nose cultures for Aspergillus flavus or fumigatus and microbiologically documented pulmonary aspergillosis were considered clinically, although not microbiologically, documented. Predisposing factors for aspergillus sinusitis during the 60 days prior to infection diagnosis were granulocyte count less than 500 microliter (mean duration, 42 days versus 14 days for sinusitis of other etiology; P less than 0.001), prolonged hospitalization (mean duration, 22 days versus 14 days for patients with nonfungal sinusitis; P less than 0.001), and prolonged antibiotic therapy (mean duration, 22 days versus 9 days; P less than 0.001). Treatment with amphotericin B was initially successful for 18 of 21 patients; however, 11 of 18 patients had infection recurrence that always developed at time of tumor exacerbation and reinstitution or intensification of chemotherapy. These findings suggest that aspergillus sinusitis in cancer patients is seen in association with prolonged neutropenia and antibiotic therapy, is amenable to therapy, but tends to recur with relapse of malignancy.

Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies.

CD44 molecules are comprised of multiple alternatively spliced forms and are associated with diverse functions such as mediation of carcinoma metastasis and T cell coactivation. To study the function of individual CD44 isoforms, we have transfected CD44 isoforms into CD44-negative Jurkat T cells and produced cloned Jurkat cell lines that are stably transfected with either a CD44 isoform containing no alternatively spliced insert (CD44H) or a CD44 variant (CD44E) containing an insert of 132 amino acids derived from exons 12, 13, and 14 of the CD44 gene. We found that neither CD44H- nor CD44E-transfected Jurkat T cells constitutively bound hyaluronan (HA), whereas PMA treatment induced Jurkat cells transfected with CD44H but not CD44E to bind HA. CD44 mAb against noninsert regions of the CD44 extracellular domain (A3D8, A1G3) and polyclonal antisera against the COOH-terminal extracellular glycosaminoglycan region of CD44H (anti-6A serum) both induced CD44H-transfected cells to bind HA, whereas only one CD44 mAb (A1G3) induced CD44E-transfected Jurkat T cells to bind HA. Studies of Jurkat cells transfected with CD44H forms with truncations of the CD44 cytoplasmic domain demonstrated that the cytoplasmic COOH-terminal 52 amino acids were critical for binding of HA to the CD44 extracellular domain. Thus, these data underscore the importance of the CD44 cytoplasmic domain in the function of the extracellular portion of CD44H, and demonstrate a role for ligation of human CD44 isoforms at multiple distinct sites in regulation of expression of CD44 binding to HA.

Nuclear magnetic resonance analysis of solution conformations in C4-V3 hybrid peptides derived from human immunodeficiency virus (HIV) type 1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies.

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.

Induction of HIVMN neutralizing antibodies in primates using a prime-boost regimen of hybrid synthetic gp120 envelope peptides.

We have tested synthetic peptides composed of Th (T1) and V3 loop B cell neutralizing determinants [SP10 MN(A)] of HIVMN gp120 and the fusogenic (F) domain of gp41 as immunogens in rhesus monkeys. After two immunizations with either HIV env peptide T1-SP10 MN(A) or F-T1-SP10 MN(A), rhesus monkey serum neutralization titers against the HIVMN isolate ranged from 1:160 to 1:1400, and in cell-cell syncytium inhibition assay ranged from 1:20 to 1:80. However, in contrast to animals immunized with T1-SP10 MN(A), animals immunized twice with F-T1-SP10 MN(A) had no rise in anti-gp120 and neutralizing antibodies with an additional immunization with F-T1-SP10 MN(A) peptide. One of 4 rhesus monkeys (18987) had anti-HIVMN antibodies that cross-neutralized divergent HIV isolates HIVIIIB and HIVRF. Serum from animal 18987 neutralized 5 of 10 HIV isolates tested, and neutralizing activity against HIVIIIB of 18987 serum was absorbed with the conserved gp120 loop V3 sequence IGPGRAF. Anti-HIV neutralizing antibodies were boosted after a 6-mo rest by 500 micrograms of T1-SP10 MN(A) in 4 of 4 animals previously immunized with T1-SP10 MN(A) and in 2 of 2 animals previously immunized with F-T1-SP10 MN(A). However, immunization after 6-mo rest of animal 18987 with 500 micrograms of T1-SP10 MN(A) peptide, although boosting anti-HIVMN neutralizing antibodies, selectively did not boost cross-neutralizing anti-HIVIIIB antibodies. Thus, synthetic peptides containing T and B cell epitopes of HIV gp120 can induce high levels of anti-HIVMN neutralizing antibodies in primates.