Genital secretions are the major source of HIV infections. Sexual transmission of HIV, though an inefficient but is the most prevalent route accounting for about 75 to 85 % of HIV infections. Virus present in the seminal components including seminal plasma, seminal leukocytes and sperm is the primary source of infection. Semen is the major vehicle for the sexual transmission of HIV-1 in adults. The ability to isolate infectious HIV from the semen and to quantify the viral burden in the form of cell-free or cell associated HIV-1 RNA in semen are important for epidemiologic and public health aspects of the epidemic. The knowledge of the effect of tropical microbicides and /or antiretrovirals on the reduction of viral load in semen is necessary for the evaluation of compounds that may be useful in prevention of sexual transmission of HIV. 

Protocol :

 To isolate HIV DNA/RNA in seminal plasma, seminal leukocytes, and   Sperm of HIV infected individuals.

Materials required : 

RPMI 1640 Medium w/L Glutamine
Ficoll-hypaque, Percoll
Ham’s F-10 Medium with L-Glutamine
Human IL-2 Medium (Boehninger Mannheim)
PHA-P (Sigma)
Percoll (sigma)
FBS (10%)
Penicillin –streptomycin solution
Haemocytometer, cover slips and Microscope

Percoll Gradient Centrifugation of Human semen sample

Preparation of stock reagents
10x Ham’s F-10: Powdered Ham’s F-10 and 1.68 g sodium bicarbonate bring to 100 ml with distilled water use once and discard or sterile filter

Prepare 1x Ham’s F-10 Medium –1ml of Ham’s F-10 (10X) +9ml of autoclaved   distilled water

Prepare Stock Percoll  (9ml of percoll +1ml of Ham’s F-10 (1X filter sterilized)
  
Preparation of 90% percoll    – Add 9ml of stock percoll
+ 1ml of Ham’s F-10 (1X) 

Preparation of 45% percoll    – Add 4.5ml of stock percoll
+ 5.5ml of Ham’s F-10 (1X)

Samples :

1. Samples were collected by masturbation into a sterile polypropylene flask after three to five days of ejaculatory abstinence and processed within 4 hours after collection.
2. The semen sample was divided into two aliquots. The first aliquot was initially submitted to semen analysis according to the World Health Organization (WHO) guidelines.
3. Following the semen analysis the second aliquot was subjected to semen processing using percoll gradient centrifugation technique at two different concentrations (45% and 90%) respectively.
4. Add equal volume of Ham’s F-10 (1X) in semen sample mix properly centrifuge at 700 rpm for 10 mins and save the supernatant which contains seminal plasma for viral load assay.
5. First overlay the pellet with 90% Percoll on the top and slowly add 1 ml of 45% Percoll centrifuge at 500g for 20 mins, after centrifugation three different fractions were obtained supernatant contains seminal plasma, middle layer contains cellular elements other than sperm (germ line cells, cellular debris, seminal leuckocytes) and the pellet. Slowly dip the pipette tip to the bottom and take only the pellet with minimum medium to obtain motile sperms.
6. Seminal plasma samples were then subjected to nucleic acid extraction using MagNa pure Compact nucleic acid isolation kit (Roche) according to the manufacturer’s instruction Nucleic acids were eluted in a 50 l of a specific buffer and stored at -70C until use and processed for viral load assay by real time PCR (Roche)
7. Sperm DNA was extracted by using sperm DNA isolation kit and DNA from seminal leuckocytes also extracted by Qiagen DNA extraction kit and further used for PCR amplification of desired gene
8. The seminal components obtained by a Percoll gradient technique were co cultured from freshly isolated peripheral blood mononuclear cells (PBMCs) co cultured with 2X 106 PBMCs from healthy donors that had been previously stimulated for 48 hours with phytohemagglutinin (PHA)-P. growth of the cell in culture was supported by RPMI 1640 media that had been supplemented with 2mM L-glutamine, 15 % heated-inactivated fetal bovine serum, 100ml/ml penicillin, 100mg/ml streptomycin and 10U/ml interleukin 2 (IL-2). The cultures were incubated at 37C in 5% CO2. The culture was maintained for 4 weeks and supernatant was collected once a week to test for the presence of p24 HIV-1 antigen by HIV-1 P24 Antigen assay kit (Perkin Elmer) Individual isolates were then collected, stored at -80C, and subsequently used to evaluate tropism and syncytium inducing activity.

Storage:

HIV samples are highly sensitive to degradation during storage at room temperature, although reverse transcriptase activity or P24 antigen levels remain relatively stable. Viral stocks are relatively stable for 6 to 12 months at -70°C.

Dr A H Bandivdekar                                                
Shivaji K. Jadhav
Assitant Director Research Fellow
National Institute for Research in Reproductive Health,
ICMR J M Street Parel Mumbai-400012.