Many of the critical events associated with HIV pathogenesis seem to occur in the first few days after infection (21
). The development of effective intervention strategies to prevent HIV transmission and AIDS requires a detailed understanding of this acute stage of infection. Among regular blood donors, periods of transient viremia in the pre-ramp-up stage of HIV infection have been reported (12
). Intermittent detection of very low levels of vDNA in the PBMC of highly exposed seronegative subjects (so-called occult infections) has been reported (45
). Similarly, occult systemic SIV infections with periods of transient viremia occur in some rhesus macaques mucosally inoculated with low doses of pathogenic SIV (24
). In the present study, we did not observe SIV transmission following infusion of 2 ml of plasma from a macaque with “blip” viremia (731 vRNA copies/ml). We also failed to transmit SIV following infusions of 40 ml of plasma collected from three macaques that had been exposed to serial intravaginal SIV challenges and had evidence of low levels of SIV DNA and RNA in tissues but failed to develop systemic plasma viremia. Although these results suggest that many occult infections, evidenced by transient blips of vRNA in plasma or viral DNA in tissue, may not be a concern with respect to blood transfusion safety, definitive conclusions in this regard cannot be made due to the limited size and scope of the present study.
Our study clearly demonstrated that a pre-ramp-up-stage plasma pool testing negative for vRNA (<3 copies/ml) and composed of plasma samples collected from six animals at least 1 week prior to the presence of measurable plasma vRNA contains infectious virus that can be transmitted to naïve macaques by i.v. inoculation. The six animals from whom this infectious pre-ramp-up-stage plasma was collected developed productive systemic infections. Moreover, it appears that the pre-ramp-up-stage virus is well adapted to replicate in the SIV-naïve host. Each recipient received 85 ml of a plasma pool containing <3 vRNA copies/ml, or <255 vRNA copies total. Nevertheless, infection was efficiently and effectively established; the animals infected with the pre-ramp-up-stage plasma had very high levels of viral replication 1 week after inoculation, and plasma vRNA levels remained well above 106 copies/ml during the 14-week observation period. These data clearly demonstrate the infectious and pathogenic potential of pre-ramp-up-stage virus and underscore the point that depending on the volume of the inoculum, even samples that test below stringent vRNA copy-per-milliliter thresholds may still transmit infection.
The plasma transfer experiments using dilutions of pools of ramp-up- and set-point-stage plasma collected from macaques after vaginal SIV inoculation demonstrated that the number of infectious virions per vRNA copy is significantly lower in set-point-stage plasma than in ramp-up-stage plasma. In fact, since each virion has 2 RNA copies, the nominal particle infectivity ratio in the ramp-up-stage plasma pool was 1 to 9 infectious units/10 virions. In marked contrast, the particle infectivity ratio in the set-point-stage plasma pool was significantly lower, at 1 to 9 infectious units/750 virions. The relatively low (1:75 to 1:750) ratio of infectious virions to total virions in set-point-stage plasma could be consistent either with the generation of less fit mutant viruses in vivo, due to the nucleotide substitution errors introduced by the SIV reverse transcriptase and host polymerases, or with the presence of antibodies that coat and neutralize a large proportion of the virions in set-point-stage plasma, or both. The hypothesis that set-point-stage virions are coated with antibodies that interfere with infectivity is consistent with the observed inactivation of the ramp-up-stage virions after they were mixed with the heat-inactivated set-point-stage plasma pool. However, we are conducting additional evaluations to determine what component(s) of set-point-stage plasma confers this activity. In this context, it is worth noting that in an early report, HIV immune globulin failed to protect chimpanzees against experimental challenge with HIV (38
) but then was clearly protective when used at a higher dose (39
). HIV immune globulin obtained from HIV-infected chimpanzees can reduce the infectivity of HIV in rhesus macaques (17
) as well as blocking simian/human immunodeficiency virus infection (41
While it may be relatively easy to explain the presence of 1 to 2 log units of noninfectious virions in set-point-phase plasma, the paucity of noninfectious virions in ramp-up-stage plasma is more difficult to explain. It is possible that although mutant genomes arise by reverse transcriptase errors during the ramp-up stage, insufficient mutations accumulate in a very fit founder virus genome during the rounds of replication between infection and ramp-up viremia to have a distinguishable effect on the fitness of the mutant virions.
A less likely hypothesis is that efficient purifying selection working through substrate competition eliminates less prolific genomes before they produce virions that reach the plasma in the short time between infection and the ramp-up stage of infection.
Differential infectivity of a virus in plasma during the acute versus the chronic stage is not unique to SIV and presumably HIV; a similar phenomenon has been reported for hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. The HCV strain H inoculum consists of serum collected 7 weeks posttransfusion from a patient in the acute stage of HCV infection, while the HCV strain F inoculum is derived from sera collected 1 year posttransfusion from a patient in the chronic stage of HCV infection (2
). The ratio of vRNA copies to infectious units is approximately 1:1 for the acute-stage strain H inoculum and >1:103
for the chronic-stage strain F inoculum (2
). In fact, as few as 20 HCV RNA copies in acute-stage serum can transmit HCV infection to a naïve chimpanzee, while HCV transmission with plasma collected after seroconversion requires 1,000-fold higher levels of HCV (18
). Similar findings of very high infectivity of ramp-up- versus set-point-stage plasma have been established for HBV, both in chimpanzees and in chimeric mice with humanized livers (20
); in the latter system, ramp-up-stage HBV serum is about 100 times more infectious than later-stage serum. As with SIV and HIV, the potential explanations for the relatively low infectivity of chronic-stage plasma in HCV infection include the presence of large numbers of defective virions or noninfectious antibody-virion immune complexes. The latter explanation is generally favored, since the results of one study suggest that a significant proportion of the HCV virions in the chronic-stage strain F inoculum exist as immune complexes (15
In human blood banking, NAT is currently used to screen blood donations for HIV, HCV, and HBV in order to prevent transfusion of blood collected during the window period between the development of infectious viremia and seroconversion. This window period includes the pre-ramp-up and ramp-up stages of infection. NAT screening was implemented in the United States and other countries in 1999 using MP screening in which 16 or more donor specimens are pooled prior to testing (8
). Prior to the use of MP-NAT, HIV was transmitted via window-stage donations at significant rates (5
). Although very rarely, HIV has been transmitted by window period blood donations that were determined to have ≤150 vRNA copies/ml even after the adoption of MP-NAT (9
). The occurrence of rare HIV transmission events by donations with no evidence of anti-HIV antibodies and very low vRNA levels is consistent with our findings. Further, our study documents transmission of infection by pre-ramp-up-stage and diluted ramp-up-stage plasma with vRNA levels below even the limit of detection of individual-donation NAT, suggesting that even individual-donation NAT, which was recently implemented in the Republic of South Africa (14
), may not be sensitive enough to interdict all HIV-infected donations. Thus, implementation of pathogen inactivation methods to sterilize blood transfusions (1
) may be required to achieve the next level of safety.
Understanding the relative infectiousness of plasma virions at different stages of HIV infection/exposure not only provides important information for assessing the safety of blood donations and donor-screening policies but may also yield significant insights into critical biological differences between transmitted virus and the virus variants that emerge during infection of the host (19
). Further studies aimed at understanding the viral phylogenetics in the plasma pool donors and recipients are under way.