Recognition and killing of Brugia malayi microfilariae by human immune cells is dependent on the parasite sample and is not altered by ivermectin treatment

Mass administration of macrocyclic lactones targets the transmission of the causative agents of lymphatic filariasis to their insect vectors by rapidly clearing microfilariae (Mf) from the circulation. It has been proposed that the anti-filarial action of these drugs may be mediated through the host immune system. We recently developed an in vitro assay for monitoring the attachment to and killing of B. malayi Mf by human neutrophils (PMNs) and monocytes (PBMCs), however, the levels of both cell to worm attachment and leukocyte mediated Mf killing varied greatly between individual experiments. To determine whether differences in an individual's immune cells or the Mf themselves might account for the variability in survival, PMNs and PBMCs were isolated from 12 donors every week for 4 weeks and the cells used for survival assays with a different batch of Mf, thereby keeping donors constant but varying the Mf sample. Results from these experiments indicate that, overall, killing is Mf-rather than donor-dependent. To assess whether ivermectin (IVM) or diethylcarbamazine (DEC) increase killing, Mf were incubated either alone or with immune cells in the presence of IVM or DEC. Neither drug induced a significant difference in the survival of Mf whether cultured with or without cells, with the exception of DEC at 2 h post incubation. In addition, human PBMCs and PMNs were incubated with IVM or DEC for 1 h or 16 h prior to RNA extraction and Illumina sequencing. Although donor-to-donor variation may mask subtle differences in gene expression, principle component analysis of the RNASeq data indicates that there is no significant change in the expression of any genes from the treated cells versus controls. Together these data suggest that IVM and DEC have little direct effect on immune cells involved in the rapid clearance of Mf from the circulation. Keywords: Brugia malayi, Ivermectin, Microfilariae, Neutrophils, Peripheral blood mononuclear cell

Evaluating a parainfluenza virus 5-based vaccine in a host with pre-existing immunity against parainfluenza virus 5.

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans

Analysis of Measles-Mumps-Rubella (MMR) Titers of Recovered COVID-19 Patients

The measles-mumps-rubella (MMR) vaccine has been theorized to provide protection against coronavirus disease 2019 (COVID-19). Our aim was to determine whether any MMR IgG titers are inversely correlated with severity in recovered COVID-19 patients previously vaccinated with MMR II. We divided 80 subjects into two groups, comparing MMR titers to recent COVID-19 severity levels. The MMR II group consisted of 50 subjects who would primarily have MMR antibodies from the MMR II vaccine, and a comparison group of 30 subjects consisted of those who would primarily have MMR antibodies from sources other than MMR II, including prior measles, mumps, and/or rubella illnesses. There was a significant inverse correlation (rs = −0.71, P < 0.001) between mumps virus titers (mumps titers) and COVID-19 severity within the MMR II group. There were no significant correlations between mumps titers and severity in the comparison group, between mumps titers and age in the MMR II group, or between severity and measles or rubella titers in either group. Within the MMR II group, mumps titers of 134 to 300 arbitrary units (AU)/ml (n = 8) were found only in those who were functionally immune or asymptomatic; all with mild symptoms had mumps titers below 134 AU/ml (n = 17); all with moderate symptoms had mumps titers below 75 AU/ml (n = 11); all who had been hospitalized and had required oxygen had mumps titers below 32 AU/ml (n = 5). Our results demonstrate that there is a significant inverse correlation between mumps titers from MMR II and COVID-19 severity

PIV5 antibodies in humans.

<p>45 human serum samples were obtained from 18–50 year old healthy individuals. (A) Comparison of anti-PIV5 and anti-MuV antibody levels. ELISA was performed on plates coated with purified PIV5 or purified MuV with sera serially diluted. PIV5 or Mumps virus specific ELISA OD₄₅₀ values were shown at 320-fold dilution for each human serum sample. (B) Titers of neutralizing antibody against PIV5 in human sera. Data for the antibody titers were the average value of duplicate wells and presented for each human sample. The white column indicates that the PIV5 nAb titer is less than 10, the limit of detection. The black column indicates that the nAb titer is equal to or higher than 10.</p

Titers of anti-PIV5 antibodies in the PIV5-vaccinated dogs.

<p>Eight dogs which had been vaccinated with live PIV5 were immunized with one dose of 8×10⁷ PFU of rPIV5-H3 viruses in 1 mL or PBS via intranasal route. The dogs were divided into two groups: two dogs received PBS; The remaining six dogs received rPIV5-H3. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and viral neutralization antibody assay (B). Data were presented as average value of duplicate wells. In the neutralization antibody assay, the white column indicates the PIV5 nAb titer is equal to or higher than 10.</p

Immune responses in the “PIV5 naïve” dogs inoculated with rPIV5-H3.

<p>The dog blood samples were collected at 0 and 21 days post infection. 4 HAU of the influenza A virus (A/Udorn/72, H3N2 subtype) were mixed with serially diluted dog sera in 96-well round-bottom plates. The hemagglutination inhibition (HAI) titer was scored as the reciprocal of the highest dilution antiserum that completely inhibits hemagglutination. The graph shows the mean value of duplicate wells for each dog. The limit of detection of the HAI titer (10) is indicated.</p

Replication of PIV5 in dogs with prior PIV5 vaccination.

<p>The nasal swabs of dogs were collected at 3 and 5 dpi. Detections of virus were performed the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050144#pone-0050144-g002" target="_blank">Fig. 2</a>. (A) RT-PCR and (B) Plaque assay.</p

Titers of anti-PIV5 antibodies in dogs without PIV5 exposure.

<p>Eight PIV5 naïve dogs were immunized with one dose of 8×10⁷ PFU of PIV5 or rPIV5-H3 viruses by intranasal route. The dogs were divided into two groups: PIV5-infected dogs and rPIV5-H3-infected dogs. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and virus neutralization antibody (nAb) assay (B). The grey columns indicate that the PIV5 nAb titer is less than 10, the limit of detection in this assay. The black columns indicate that the nAb titer is equal to or higher than 10.</p

Replication of PIV5 in dogs without PIV5 exposure.

<p>The nasal swabs of dogs were collected at 3 and 5 days post infection, and placed into a vial containing 0.5 mL of DMEM with 2% FBS. (A) Detection of virus with RT-PCR. (B) Detection of virus with plaque assay. Swab samples were examined by plaque assay on BHK21 cells. Two replicates for each serially diluted swab sample (1∶10⁰ to 1∶10²) were used in the assay.</p