Abstract

The survival of 7 of 8 patients with Ebola virus (EBOV) infection after transfusions of convalescent-phase blood during a 1995 outbreak of EBOV infection is frequently cited as evidence that passive immunotherapy is a viable treatment option. To test whether whole-blood transfusions were more efficacious than passively administered immunoglobulins or monoclonal antibodies, we transfused convalescent-phase blood from EBOV-immune monkeys into naive animals shortly after challenge with EBOV. Although passively acquired antibody titers comparable to those associated with effective vaccination were obtained, all monkeys that had received transfusions succumbed to infection concurrently with control monkeys. These data cast further doubt on the value of passive immunotherapy for the treatment of EBOV infection.

Ebola virus (EBOV) causes severe hemorrhagic fever with high mortality rates among humans and nonhuman primates [1]. Currently, there are no vaccines or therapies available for human use. Treatment is primarily supportive management and palliative care. The recurrence of outbreaks of EBOV infection in central Africa, along with the potential use of EBOV as a weapon [2] and the recent death of a Russian scientist after accidental exposure to EBOV [3], underscores the need for medical countermeasures and prophylaxis. The treatment of laboratory-acquired infection by use of convalescent-phase serum from EBOV-infected patients in 1976 [4] and the reported survival of 7 of 8 EBOV-infected patients after transfusions of convalescent-phase blood during the 1995 outbreak in Kikwit, Democratic Republic of the Congo [5], are frequently cited as evidence that passive immunotherapy is a viable treatment option. Although those involved with the Kikwit study [5, 6] acknowledged that a number of factors other than passively administered antibody might have been associated with the enhanced survival rates observed among these patients, this approach continues to draw advocates of further development, despite a preponderance of data documenting the failure of passive immunotherapy in the treatment of Ebola hemorrhagic fever (EHF) in primates. In the present study, we tested the hypothesis that whole blood (analogous to that obtained from EBOV-immune donors in Kikwit) might confer some protection to EBOV-infected recipients.

Materials and Methods

To obtain immune monkey blood, 3 rhesus macaques that had survived challenge with EBOV 4–5 years previously were rechallenged with a high dose (1000 pfu) of infectious Zaire EBOV (ZEBOV). None of these donor animals developed clinical signs of EHF, and all were aviremic after rechallenge. Of these 3 animals, 1 had survived a primary challenge with mouse-adapted ZEBOV in 1997, which is thought to be attenuated in nonhuman primates, and a subsequent challenge with wild-type ZEBOV in 1999 [7]. A second monkey had been vaccinated with a girradiated whole-virion vaccine and survived subsequent challenge with ZEBOV [8], and the third monkey had been one of the rare, untreated survivors of virulent ZEBOV infection (P.B.J., unpublished data). Blood was obtained from the donor monkeys 30 days after rechallenge with 1000 pfu of ZEBOV. Anti-EBOV IgG ELISA titers for all 3 donor animals were 1:100,000 (table 1).

Table 1

ELISA antibody titers against Ebola virus (EBOV) in rhesus macaques before and after challenge with Zaire EBOV (ZEBOV).

Table 1

ELISA antibody titers against Ebola virus (EBOV) in rhesus macaques before and after challenge with Zaire EBOV (ZEBOV).

To maximize the likelihood of a beneficial effect, recipient monkeys received transfusions immediately after ZEBOV exposure. In contrast, during the Kikwit outbreak, patients received transfusions 4–15 days after the onset of clinical symptoms; transfusion volumes were 150–450 mL per patient (∼2.1–6.4 mL/kg). In the animal model, 4 monkeys received 6 mL/kg citrate phosphate adenine–treated whole blood from convalescent-phase donors. Two of the recipients received a second transfusion (6 mL/kg) on day 3 after challenge with ZEBOV, and the other 2 recipients received the second transfusion (6 mL/kg) on day 4 after challenge. Two ZEBOV-challenged control animals also were used. One control animal was an EBOV-naive recipient that received transfusion in parallel with whole blood from an EBOV-naive donor monkey immediately after challenge with ZEBOV and again on day 3 after challenge; the second control animal did not receive convalescent-phase blood after challenge with ZEBOV.

Research was conducted in compliance with the AnimalWelfare Act and other federal statues and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals [9]. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (Rockville, MD).

Results

For the donor and recipient animals, pre- and postchallenge ELISA titers against EBOV are presented in table 1. In brief, 3 of the 4 recipient monkeys had passive ELISA titers of 1:100 at day 3 (recipient 2) or day 4 (recipients 3 and 4) after challenge with ZEBOV, and the remaining monkey (recipient 1) had ELISA titers of 1:3200 at day 3. Recipient monkeys 1–4 and both of the ZEBOV-challenged control monkeys developed clinical signs consistent with EHF and became viremic during the course of infection, with ⩾5.0 log10 pfu/mL plasma by day 7 (figure 1). Two of the recipient monkeys were killed in extremis late on day 8 after challenge, while the other 2 recipients and both positive control monkeys died on day 9. The mean time to death for a cohort (n=23) of ZEBOV-positive control rhesus monkeys inoculated with the same isolate, with the same dose and route, was 8.3 days (T.W.G., unpublished data) [8].

Figure 1

Plasma viremia levels of rhesus macaques after challenge with Zaire Ebola virus (ZEBOV). Control 1 was an EBOV-naive recipient animal that received transfusion in parallel with whole blood from an EBOV-naive donor monkey immediately after challenge with ZEBOV and again on day 3 after challenge. Control 2 did not receive convalescent-phase blood after challenge with ZEBOV.

Figure 1

Plasma viremia levels of rhesus macaques after challenge with Zaire Ebola virus (ZEBOV). Control 1 was an EBOV-naive recipient animal that received transfusion in parallel with whole blood from an EBOV-naive donor monkey immediately after challenge with ZEBOV and again on day 3 after challenge. Control 2 did not receive convalescent-phase blood after challenge with ZEBOV.

Discussion

Our results show no beneficial effect from the transfusion of convalescent-phase blood from rhesus macaques immune to ZEBOV infection, in a controlled experiment in which the therapeutic regimen was adjusted for maximum beneficial effect. The results of this study support the idea suggested by the authors of the Kikwit study [5] that enhanced survival was unlikely to be related to the presence of EBOV-specific antibodies in donor blood. The present study did not use blood from animals recently in the convalescent phase of infection (in contrast, the human blood used in the Kikwit study was obtained 37–65 days after patients were discharged from the hospital); however, it is unlikely that protective antibodies would exist in the human blood that were absent in the immune monkey blood used here.

Furthermore, the suggestion that antibody titers may have no direct role in conferring protection is based on our observation that, in this study, the passive ELISA titers in 1 of the 4 recipient animals at day 3 after challenge (1:3200) was equivalent to or higher than prechallenge ELISA titers in animals protected against lethal ZEBOV challenge by immunization with either a recombinant adenovirus-based EBOV vaccine [10] or a recombinant vesicular stomatitis virus (VSV)-based EBOV vaccine [11]. In addition, passive ELISA titers in the other 3 recipient animals at day 3 or 4 after challenge (1:100) were nearly as high as prechallenge ELISA titers in half the animals (1:320) protected against ZEBOV by the VSV-based EBOV vaccine [11]. In fact, the same ELISA assay for EBOV IgG was used in all of these studies, so comparisons of the results are relevant. With regard to protective efficacy, the questionable relevance of anti-EBOV ELISA titers was also mentioned in a recent study comparing 2 EBOV vaccines [12] that differed dramatically in protective efficacy but that elicited essentially identical results for anti-EBOV ELISA titers.

The findings of the current study are consistent with those of previous studies showing that passive immunization of nonhuman primates with anti-ZEBOV antibodies has little therapeutic utility. These study results include the following: (1) passive immunization of cynomolgus macaques with high-titer anti-EBOV equine immunoglobulin was unable to protect animals from a lethal ZEBOV challenge [13, 14]; (2) passive immunization of 4 rhesus macaques with high-titer anti-EBOV equine immunoglobulin failed to protect any of the animals from a lethal challenge and did not even delay death (P.B.J., unpublished data); and (3) passive transfer of a neutralizing human monoclonal antibody that completely protected guinea pigs against ZEBOV infection [15] failed to protect rhesus macaques against a lethal ZEBOV challenge [16].

The severity of EBOV infection and the lack of effective therapeutic solutions argue for sustained investment in all promising approaches. However, the results of the present study, which used immune primate blood, joins the preponderance of published study results suggesting that immunotherapy will not be a shortcut to the solution. Given these discouraging results and the risks of transmitting infection, whole-blood transfusions, even under desperate epidemic conditions, seem unwarranted.

Acknowledgments

We thank Denise Braun, Carlton Rice, and James Stockman for technical assistance and assistance with animal care.

Supplement sponsorship. This article was published as part of a supplement entitled “Filoviruses: Recent Advances and Future Challenges,” sponsored by the Public Health Agency of Canada, the National Institutes of Health, the Canadian Institutes of Health Research, Cangene, CUH2A, Smith Carter, Hemisphere Engineering, Crucell, and the International Centre for Infectious Diseases.

References

1.
Sanchez
A
Geisbert
TW
Feldmann
H
Knipe
DM
Howley
PM
Griffin
DE
, et al.  . 
Filoviridae: Marburg and Ebola viruses
Fields virology
 , 
2007
Philadelphia
Lippincott, Williams & Wilkins
(pg. 
1409
-
48
)
2.
Borio
L
Inglesby
T
Peters
CJ
, et al.  . 
Hemorrhagic fever viruses as biological weapons: medical and public health management
JAMA
 , 
2002
, vol. 
287
 (pg. 
2391
-
405
)
3.
International Society for Infectious Diseases
Ebola, lab accident death—Russia (Siberia)
ProMed archive 20040522.1377
  
Available at: http://www.promedmail.org. Accessed 22 May 2004
4.
Emond
RT
Evans
B
Bowen
ET
Lloyd
G
A case of Ebola virus infection
Br Med J
 , 
1977
, vol. 
2
 (pg. 
541
-
4
)
5.
Mupapa
K
Massamba
M
Kibadi
K
, et al.  . 
Treatment of Ebola hemorrhagic fever with blood transfusions from convalescent patients
J Infect Dis
 , 
1999
, vol. 
179
 
(Suppl 1)
(pg. 
S18
-
23
)
6.
Sadek
RF
Khan
AS
Stevens
G
Peters
CJ
Ksiazek
TG
Ebola hemorrhagic fever, Democratic Republic of the Congo, 1995: determinants of survival
J Infect Dis
 , 
1999
, vol. 
179
 
(Suppl 1)
(pg. 
S24
-
7
)
7.
Bray
M
Hatfill
S
Hensley
L
Huggins
JW
Haematological, biochemical and coagulation changes in mice, guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus
J Comp Pathol
 , 
2001
, vol. 
125
 (pg. 
243
-
53
)
8.
Geisbert
TW
Pushko
P
Anderson
K
Smith
J
Davis
KJ
Jahrling
PB
Evaluation in nonhuman primates of vaccines against Ebola virus
Emerg Infect Dis
 , 
2002
, vol. 
8
 (pg. 
503
-
7
)
9.
Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources Commission of Life Sciences, National Research Council
Guide for the care and use of laboratory animals
1996
Washington, DC
National Academy Press
10.
Sullivan
NJ
Geisbert
TW
Geisbert
JB
, et al.  . 
Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates
Nature
 , 
2003
, vol. 
424
 (pg. 
681
-
4
)
11.
Jones
SM
Feldmann
H
Stroher
U
, et al.  . 
Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses
Nat Med
 , 
2005
, vol. 
11
 (pg. 
786
-
90
)
12.
Sullivan
NJ
Geisbert
TW
Geisbert
JB
, et al.  . 
Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs
PLoS Med
 , 
2006
, vol. 
3
 (pg. 
865
-
73
)
13.
Jahrling
PB
Geisbert
J
Swearengen
JR
, et al.  . 
Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses
Arch Virol Suppl
 , 
1996
, vol. 
11
 (pg. 
135
-
40
)
14.
Jahrling
PB
Geisbert
TW
Geisbert
JB
, et al.  . 
Evaluation of immune globulin and recombinant interferon-α2b for treatment of experimental Ebola virus infections
J Infect Dis
 , 
1999
, vol. 
179
 
(Suppl 1)
(pg. 
S224
-
34
)
15.
Parren
PWHI
Geisbert
TW
Maruyama
T
Jahrling
PB
Burton
DR
Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody
J Virol
 , 
2002
, vol. 
76
 (pg. 
6408
-
12
)
16.
Oswald
WB
Geisbert
TW
Davis
KJ
, et al.  . 
Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys
PLoS Pathog
 , 
2007
, vol. 
3
 (pg. 
62
-
6
)
Potential conflicts of interest: none reported.
Presented in part: Filoviruses: Recent Advances and Future Challenges, International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba, Canada, 17–19 September 2006.
Financial support: Defense Threat Reduction Agency and the Medical Chemical/Biological Defense Research Program, US Army Medical Research and Material Command (project 02-4-4J-084). Supplement sponsorship is detailed in the Acknowledgments.
Opinions, interpretations, conclusions, and recommendations expressed in this article are those of the authors and are not necessarily endorsed by the National Institute of Allergy and Infectious Diseases, National Institutes of Health; the US Army; or the Association for Assessment and Accreditation of Laboratory Animal Care International.