Demographics and clinical characteristics of hospitalised patients under investigation for COVID-19 with an initial negative SARS-CoV-2 PCR test result.

BACKGROUND: The COVID-19 pandemic is placing abnormally high and ongoing demands on healthcare systems. Little is known about the full effect of the COVID-19 pandemic on diseases other than COVID-19 in the South African setting. OBJECTIVE: To describe a cohort of hospitalised patients under investigation for SARS-CoV-2 that initially tested negative. METHODS: Consecutive patients hospitalised at Khayelitsha Hospital from April to June 2020, whose initial polymerase chain reaction test for SARS-CoV-2 was negative were included. Patient demographics, clinical characteristics, ICD-10 (International Statistical Classification of Diseases and Related Health Problems 10th Revision) diagnosis, referral to tertiary level facilities and ICU, and all-cause in-hospital mortality were collected. The 90-day re-test rate was determined and comparisons were made using the χ2-test and the independent samples median test. RESULTS: Overall, 261 patients were included: median age 39.8 years, 55.6% female (n = 145). Frequent comorbidities included HIV (41.4%), hypertension (26.4%), and previous or current tuberculosis (24.1%). Nine (3.7%) patients were admitted to ICU and 38 (15.6%) patients died. Ninety-three patients (35.6%) were re-tested and 21 (22.6%) were positive for SARS-CoV-2. The top primary diagnoses related to respiratory diseases (n = 82, 33.6%), and infectious and parasitic diseases (n = 62, 25.4%). Thirty-five (14.3%) had a COVID-19 diagnostic code assigned (26 without microbiological confirmation) and 43 (16.5%) had tuberculosis. Older age (p = 0.001), chronic renal impairment (p = 0.03) and referral to higher level of care (all p < 0.001; ICU p = 0.03) were more frequent in those that died. CONCLUSION: Patients with tuberculosis and other diseases are still presenting to emergency centres with symptoms that may be attributable to SARS-CoV-2 and requiring admission. Extreme vigilance will be necessary to diagnosis and treat tuberculosis and other diseases as we emerge from the COVID-19 pandemic.

Long lived multi-isotype anti-HIV antibody responses following a prime-double boost immunization strategy.

Despite decades of work, an effective HIV vaccine remains elusive. In an effort to elicit protective immunity, investigators have sought to define vaccines able to elicit durable HIV-specific B-cell and T-cell activities. Additionally, vaccines are sought which can induce antibodies of a variety of isotypes, as each isotype possesses unique attributes in terms of opsonization, Fc receptor binding capacity, complement fixation and location. One prominent new vaccine strategy, applied to numerous distinct antigenic systems is the prime boost-regimen, with DNA, vaccinia virus (VV), and/or purified recombinant protein. To examine the durability, location and isotype distribution of responses induced by prime-boost regimens, we tested successive immunizations with DNA, VV and protein (D-V-P), comparing three forms of protein inoculations: (i) purified protein administered intramuscularly with complete Freunds adjuvant, (ii) purified protein administered intranasally, and (iii) purified protein conjugated to oxidized mannan, administered intranasally. We found that all three protocols elicited serum antibodies of multiple isotypes, with serum IgA being most prominent among mice immunized with mannan-conjugated protein. All D-V-P protocols, regardless of protein form or route, also elicited antibody responses at mucosal surfaces. In bronchoalveolar lavage, a tendency toward IgA production was again most prominent in mice boosted with the protein-mannan conjugate. Both B-cell and T-cell responses were sustained for more than 1 year post-immunization following each form of vaccination. Contemporaneous with long-lasting serum and mucosal antibodies were antibody forming cells in the bone marrow of primed animals. Results highlight the D-V-P vaccination strategy as a promising approach for attaining durable, multi-isotype B-cell and T-cell activities toward HIV.

Subcutaneous administration of a recombinant vaccinia virus vaccine expressing multiple envelopes of HIV-1.

A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors. Recombinant vaccinia virus is a highly effective vaccine vector, with demonstrated capacity to protect animals from various viral pathogens, including rabies. Unlike many other candidate vaccine vectors, vast human experience exists with the parenteral smallpox vaccine. However, consideration of recombinant vaccinia virus as a modern vaccine is complicated by the relatively high prevalence of immunocompromised persons compared to such prevalence 4 or more decades ago (when smallpox vaccination was still routine). Administering vaccine by the subcutaneous (SQ) route, rather than the traditional scarification route, could address these concerns. SQ administration could prevent transmission of vaccinia virus to potentially vulnerable persons; it could also avoid the most common adverse events, which are cutaneous in nature. However, previous studies suggest that elicitation of immune response against passenger gene products following SQ administration requires development of a superficial pox lesion, defeating the intention of SQ administration. This is the first report to demonstrate that SQ administration of recombinant vaccinia virus does elicit immune response to the passenger protein in the absence of a cutaneous pox lesion. Results further show that a multi-envelope HIV vaccine can elicit antibody responses toward heterologous HIV-1 not represented by primary sequence in the vaccine. These findings have global implications because they support the consideration of recombinant vaccinia virus as a valuable HIV vaccine vector system.

Limited breadth of a T-helper cell response to a human immunodeficiency virus envelope protein.

Single-envelope human immunodeficiency virus (HIV) vaccines have been studied for more than a decade, with some successes in homologous challenge experiments in nonhuman primates but with no clear successes in clinical trials. To gain insight into the breadth of the immunity elicited by such vaccines, we have dissected the T-helper cell response of C57BL/6 mice to an individual, molecularly cloned envelope protein. Here, we report that T-helper cells responsive to HIV type 1 1035 envelope are very highly restricted in C57BL/6 animals: seven different hybridomas recovered from five separate mice recognized the same peptide, PKVSFEPIPIHYCAP, located in the C2 region of gp120. Three of these hybridomas were tested on a natural variant of the peptide but failed to respond. A more extensive analysis of whole splenic populations from other C57BL/6 mice immunized with the 1035 envelope reproducibly confirmed that the gp120-specific T-helper response was almost exclusively focused on a single epitope. We conclude that single-envelope vaccines may frequently fail to provoke an immune response sufficiently diverse to recognize variant sequences among circulating HIV. The results encourage the inclusion of more than one envelope in future vaccines to enhance the potential diversity and respective surveillance capacities of responding T-helper cell populations.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing.

The spectrum of immunogenic epitopes presented by the H2-IA(b) MHC class II molecule to CD4(+) T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Valpha and Vbeta chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences ( approximately 20-80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

T cell immunotherapeutic populations control viral infections in bone marrow transplant recipients.

Immunotherapies designed to prevent infection serve as an increasingly important adjunct to bone marrow transplantation (BMT). T cell immunotherapies are particularly useful for the control of virus infections, provided that T cell populations are free of graft-vs-host (GVH) activity. In this review, we describe positive and negative selection methods with which donor T cell populations devoid of GVH activity can be prepared for transfer to the immunodeficient BMT recipient. The support of patients with T cell immunotherapies may ultimately revolutionize BMT, elevating the procedure from a salvage to a front-line treatment strategy for otherwise fatal disorders.

Multi-envelope HIV vaccine safety and immunogenicity in small animals and chimpanzees.

A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.

A novel vaccine regimen utilizing DNA, vaccinia virus and protein immunizations for HIV-1 envelope presentation.

Recombinant DNA and vaccinia virus (VV) vectors that express envelope (Env) proteins of the human immunodeficiency virus (HIV) have each been prominently utilized in vaccine development. These two vectors (termed DNA-Env and VV-Env) are attractive vaccine candidates due to their abilities to elicit both cytotoxic T-lymphocyte and B-cell responses. Our previous work demonstrated that DNA-Env primed animals, that were relatively unresponsive to DNA-Env boosters, could be immunized with VV-Env to yield more than a 100-fold increase in antibody responses. Here we show: (1) results with an optimized vaccine regimen that primes with DNA-Env, boosts with VV-Env, and re-boosts with purified Env proteins, (2) enhanced responses with 8 rather than 16 week intervals between VV-Env and protein immunizations, and (3) the failure of single Env vaccines to reproducibly elicit responses toward heterologous Env, regardless of the vaccination regimen utilized. Results encourage the use of poly-Env vaccine cocktails administered via DNA/VV/protein regimens in future non-human primate and clinical studies.

Effect of methoprene and 20-hydroxyecdysone on vitellogenin production in cultured fat bodies and backless explants from unfed female Dermacentor variabilis.

The effect of 20-hydroxyecdysone (20HE) and the juvenile hormone analogue methoprene (JHA) on vitellogenin (Vg) production in fat body organ cultures and backless explants of unfed female Dermacentor variabilis was measured. An indirect double antibody enzyme linked immunosorbent assay (ELISA) was developed using a monoclonal antibody that recognized a 98 kDa subunit of Vg and a Vg specific polyclonal antibody made against vitellin (Vn). Peak Vg titers in culture medium from fat body cultures treated with 0.1 &mgr;M 20HE or 1 &mgr;M 20HE were 24 ng/ml and 20 ng/ml respectively. In culture medium from backless explants treated with 0.1 &mgr;M 20HE or 1 &mgr;M 20HE, peak Vg titers were 36 ng/ml and 26 ng/ml, respectively. JHA produced only a slight increase in Vg titers that was statistically different from Vg titers produced by 20HE but was not statistically different from hormone-free controls. These results support the conclusion that Vg production in fat body trophocytes of D. variabilis is regulated by 20HE.

Eliciting HIV-1 envelope-specific antibodies with mixed vaccinia virus recombinants.

Recombinant vaccinia virus (VV) vectors that express the envelope (Env) protein of the human immunodeficiency virus-type 1 (HIV-1) have been previously shown to elicit HIV-specific cytotoxic T-lymphocyte (CTL) and weak antibody responses in non-human primate studies and clinical trials. In first clinical trials, single Env proteins were presented to the immune system by VV recombinants and other vectors, but individuals were not protected against later exposures to heterologous HIV. It is likely that the generation of protective immune responses against diverse HIV will require that vaccines encompass proteins from not just one, but multiple distinct HIV isolates. Here is described the simple construction of numerous new VV, each expressing a unique, truncated, Env protein (VVenv). Mouse experiments were performed to evaluate the ability of these VVenv to elicit immune responses. HIV-1-specific antibodies appeared within one month following one intraperitoneal inoculation of mice with single or mixed VVenv, reaching plateau levels by 4 months. The magnitude of antibody production was poor at the dose of 10(5) p.f.u. VVenv per animal, but improved with increasing doses of VVenv up to 10(7) p.f.u. per animal. The subcutaneous route of VV immunization, previously proven safe in human trials, was also effective for administering VVenv. These results highlight the strengths of recombinant VV constructs as vehicles for the presentation of multiple HIV-1-Env proteins to the naive immune system.

Eliminating graft-versus-host potential from T cell immunotherapeutic populations.

It is common knowledge that donor T cells are responsible for graft-versus-host disease (GVHD) following bone marrow transplantation (BMT), yet GVHD remains a grave threat to transplant patients. The donor marrow can be purged of T cells to reduce this danger, but the risks of viral infections, tumor relapse and graft rejection are then increased. Here we describe a method that may be used to provide BMT patients with T cell immunotherapeutic populations responsive to foreign antigens, but unresponsive to host HLA. The method involves the culture of donor T cells with host-derived B lymphoblastoid cell lines (BLCL). During culture, the T cells are activated by the mismatched host HLA. Activated cells are subsequently removed by fluorescence-activated cell sorting. Criteria for removal include cell size and the expression of multiple T cell activation antigens on cell membranes. After the procedure, T cell populations retain helper and cytotoxic T cell responses against foreign antigens, but are specifically devoid of responses to host HLA. This technique offers a promising method for providing BMT patients the benefits of T cell immunity without the consequences of GVHD.

Purification and characterization of lysozyme from hemolymph of Heliothis virescens larvae.

Lysozyme is an important antibacterial protein in the insect defense system. Lysozyme was isolated from hemolymph of Heliothis virescens larvae using gel filtration and ion-exchange chromatography. Heliothis lysozyme had a molecular mass of 16,000 daltons by SDS-PAGE. Using acid gel electrophoresis, Heliothis lysozyme migrated faster than egg white lysozyme. The pI of Heliothis lysozyme was estimated as greater than 9.5. Heliothis lysozyme had specific bactericidal activity against three Gram-positive bacteria but no activity against Escherichia coli. The bactericidal activity was stable at 100 degrees C at pH 3.0 after 60 min incubation, but was labile at 100 degrees C at pH 6.8 after 60 min incubation. Heliothis lysozyme was an inducible protein that increased 9 times when comparing unvaccinated with vaccinated larvae. Lysozyme from H. virescens was more similar in molecular mass, heat sensitivity and pH sensitivity to lysozyme isolated from Galleria mellonella and Bombyx mori than to lysozyme isolated from Hyalophora cecropia.

Formation of pores in Escherichia coli cell membranes by a cecropin isolated from hemolymph of Heliothis virescens larvae.

The insect humoral defense system produces antibacterial peptides called cecropins. Cecropins were initially isolated from Hyalophora cecropia pupae and have since been isolated and identified in various insects. In this study, we have isolated and identified a cecropin from Heliothis virescens larvae. Rabbit IgG were raised against synthetic cecropin B. Affinity chromatography with the rabbit anti-(cecropin B) IgG was used to isolate a cecropin from hemolymph of H. virescens larvae. Acid gel electrophoresis followed by a bacterial-overlay analysis showed that Heliothis cecropin is a basic peptide of low molecular mass with bactericidal activity against Escherichia coli K12 D31. Heliothis cecropin is therefore analogous to synthetic cecropin B. One unresolved issue concerning cecropins and other antibiotic peptides is the mode of action by which they kill bacteria. By means of electron microscopy and immunocytochemistry with gold-labeled rabbit anti-cecropin IgG, binding of purified and synthetic cecropin to the cell membranes of E. coli K12 D31 cells was observed. Small lesions in the cell membrane were seen that had a diameter of 9.6 nm and internal pore of 4.2 nm. The Heliothis cecropin was found to be a pore-forming molecule that causes lesions in the cell membrane of E. coli K12 D31. The lesions lead to leakage of cytoplasmic contents and death of bacteria.

Induction of cecropin-like and attacin-like antibacterial but not antiviral activity in Heliothis virescens larvae.

Inducible cecropin-like and attacin-like proteins were isolated from immune hemolymph obtained from vaccinated Heliothis virescens larvae. The attacin-like protein had a molecular weight of approximately 25,000 daltons and was not dialyzable. The cecropin-like peptide had an estimated molecular weight of 6,000-7,000 daltons and was dialyzable, heat-stable and sensitive to trypsin digestion. The cecropin-like peptide showed bactericidal activity against Escherichia coli and Enterobacter cloacae, and the attacin-like protein showed bactericidal activity against E. coli. The immune hemolymph was bactericidal against E. coli, E. cloacae and Pseudomonas aeruginosa. Ultrastructural cell envelope damage to E. coli, produced by the immune hemolymph, was observed by scanning electron microscopy. No antiviral activity by the inducible cecropin-like and attacin-like proteins was detected against herpes simplex virus-1 and the vesicular stomatitis virus.

Acute phase reactants in neonatal bacterial infection.

The C-reactive protein (CRP) level was evaluated in 142 infants requiring investigation for suspected infection. After excluding two neonates because of incomplete data, there remained 140 neonates, of whom 16 had septicemia. Fifteen of 16 had increased CRP levels. The CRP value was not elevated in any baby (n = 5) who had positive blood cultures for Staphylococcus epidermidis, all of whom had an uneventful clinical course. The CRP level was elevated in all six babies with meconium-aspiration syndrome, but was normal in five infants whose viral cultures were positive. Ninety-nine percent of uninfected babies had normal CRP values. Overall, CRP was a valuable test for diagnostic confirmation of bacterial infection. Elevated CRP level was always accompanied by at least one abnormality in the other tests performed. Although the study was not intended to predict clinical onset of bacterial disease, our results suggest that the CRP level, because of a high negative predictive value, may be useful in ruling out bacterial infection.

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.