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Animal Models and Retroviral Vaccines Section, Immune Biology of Retroviral Infection Section
Biostatistics and Data Management Section, National Cancer Institute
Biodefense Clinical Research Branch, Office of Clinical Research, National Institute of Allergy and Infectious Diseases
Division of Viral Products, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda
Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick
Southern Research Institute, Frederick, Maryland
It is unknown whether smallpox vaccination would protect human immunodeficiency virus type 1 (HIV-1)infected individuals, because helper CD4+ cells, the targets of HIV-1 infection, are necessary for the induction of both adaptive CD8+ cell and B cell responses. We have addressed this question in macaques and have demonstrated that, although smallpox vaccination is safe in immunodeficient macaques when it is preceded by immunization with highly attenuated vaccinia strains, the macaques were not protected against lethal monkeypox virus challenge if their CD4+ cell count was <300 cells/mm3. The lack of protection appeared to be associated with a defect in vaccinia-specific immunoglobulin (Ig) switching from IgM to IgG. Thus, vaccination strategies that bypass CD4+ cell help are needed to elicit IgG antibodies with high affinity and adequate tissue distribution and to restore protection against smallpox in severely immunocompromised individuals.
Large-scale smallpox vaccination with live vaccinia virus eradicated smallpox worldwide [1, 2]. Smallpox vaccination was halted in the 1970s, because its adverse effects outweighed its benefits in the absence of a smallpox threat. The deliberate introduction of smallpox through terrorist acts could therefore have serious consequences, because, at present, herd immunity is considerably reduced. Recently, the US government reintroduced vaccination for the armed forces and a portion of the civilian population [3], and concerns have arisen, because the number of immunocompromised people, who are potentially at risk for adverse effects of Dryvax, has increased because of the HIV pandemic, aggressive chemotherapy in cancer treatment, and organ transplantation.
We previously modeled, in immunocompromised macaques, smallpox-vaccination regimens for patients with AIDS and demonstrated that immunization with a highly attenuated poxvirus, such as NYVAC [4], decreases the size and hastens the resolution of Dryvax-induced skin lesions in macaques with AIDS [5]. However, we did not assess whether this prime-boost vaccination strategy would protect the macaques against lethal poxvirus challenge. The combination of the highly attenuated modified vaccinia virus Ankara (MVA) [6] followed by Dryvax has recently been shown to fully protect immunocompetent macaques against a lethal intravenous monkeypox virus challenge in a model that was developed as a surrogate model for human smallpox [7, 8]. We investigated the ability of Dryvax alone or in combination with the attenuated vaccinia virus and MVA to protect macaques that had moderate to severe depletion of CD4+ cells against the same monkeypox virus challenge. We confirm that previous NYVAC immunization improves the safety profile of Dryvax [5], and we demonstrate that the same holds true for MVA. Importantly, however, we also demonstrate that none of these vaccine regimens protect macaques that have a severe depletion of CD4+ cells (<300 cells/mm3) against monkeypox virus challenge. This lack of protection was associated with the inability of the macaques to switch the subclass of vaccinia-neutralizing immunoglobulin from IgM to IgG, likely because of insufficient CD4+ cell helper function.
MATERIALS AND METHODS
Immunization and monkeypox virus challenge.
Twenty-five colony-bred rhesus macaques (Macaca mulatta), obtained from either Covance Research Products or Laboratory Animal Breeders and Services of Virginia, were housed and handled in accordance with the standards of the American Association for the Accreditation of Laboratory Animal Care. All macaques were vaccinated with Dryvax by scarification, as described elsewhere [5]. Groups 1 and 2 received 2 inoculations of 108 pfu of MVA or NYVAC, 4 weeks apart, and Dryvax at 6 months (figure 1B). Skin lesions were photographed and measured every 2 days after the administration of Dryvax. Monkeypox virus (strain Zaire 79) was administered by the intravenous route, at a dose of 5 × 107 pfu, either 1 or 6 months after Dryvax vaccination.
Measurement of vaccinia-neutralizing antibodies.
Plasma samples from monkeys in groups 18 were collected a few weeks after vaccination with Dryvax. All plasma samples were heat inactivated (for 30 min at 56°C) and were evaluated for the presence of vaccinia-neutralizing antibodies by use of an assay based on the expression of a reporter gene, -galactosidase (-Gal) [9].
A recombinant vaccinia virus, vSC56, expressing -Gal under the control of a synthetic early/late promoter [10] was used to develop a neutralization assay based on a single-round infection of HeLa cells (CCL-2; American Type Culture Collection). This is a rapid (24-h), high-throughput assay that has been shown to have similar sensitivity to the classic plaque-reduction neutralization test [9]. Each assay included, as a positive control, US Food and Drug Administration Standard Reference Vaccinia Immunoglobulin (obtained from Dynport vaccine company and divided into vials at the Center for Biologics Evaluation and Research). Negative controls included plasma from unvaccinated macaques and albumin. Four serial dilutions of each sample of monkey plasma were preincubated with vSC56 virus for 60 min at 37°C and then dispensed into 96-well round-bottom plates that contained 1 × 105 HeLa cells/well (5 replicates/antibody dilution). Plates were incubated at 37°C in a humidified CO2 incubator for an additional 16 h. Cells were then lysed with the detergent IGEPAL CA630 (Sigma). During the second stage of the assay, the enzymatic activity of -Gal in each well was measured by use of 96-well Immunlon 2 plates (Thermo Labsystems). Each plate included a standard curve of a recombinant -Gal enzyme (Roche). Chlorophenol red -D-galactopyranoside monosodium salt substrate (Roche) was added to all wells at room temperature in the dark for 30 min, and the enzymatic reaction was stopped with 1 mol of Na2CO3 solution. The optical density was determined at 575 nm by use of an ELISA reader. Optical density readings were transferred to Microsoft Excel 2000 for further analysis. The -Gal standard curves were used to convert optical-density values into -Gal activity per experimental or control group (in milli-units per milliliter). The -Gal activity of each experimental group (virus mixed with a given dilution of test plasma) was expressed as the percentage of -Gal activity in the virus-only control wells. Microsoft Excel 2000 was used to plot the percentage of control values for the serial dilutions of each plasma-versus-log dilution. The equation of each curve was used to calculate the ID50.
DNA extraction and real-time polymerase chain reaction (PCR).
DNA was extracted from whole blood and from throat swabs by use of the QIAamp DNA mini extraction kit (Qiagen), in accordance with the manufacturer's instructions. DNA was extracted from 100 or 200 L of whole blood that had been drawn by femoral venipuncture into tubes that contained EDTA. The red blood cells were lysed before DNA extraction by storing the whole blood at -80°C overnight, and the final DNA pellet was air dried and resuspended in 100 L of sterile hydration solution (10 mmol/L Tris and 0.1 mmol/L EDTA [pH 8]). Throat swabs were collected by use of sterile cotton-tipped applicators and frozen, at -80°C, until they were processed. Swab homogenates were obtained by adding 1 mL of PBS, subjecting them to vortex, and then conducting 3 cycles of freezing/thawing and sonication for 15 s each cycle. DNA was extracted from 100 L of the homogenate, as described above. DNA samples were stored at -20°C until they were assayed.
Real-time PCR was done with the LightCycler (Roche) by use of a panorthopox virus assay, as described elsewhere [11]. Briefly, the oligonucleotide primers and a minor groove binder (MGB) protein containing a TaqMan probe were selected from conserved regions of the orthopoxviral hemagglutinin gene; their sequences have been published elsewhere [11]. The final concentrations of the components used in the 20-L reaction were as follows: 1× PCR reaction buffer (50 mmol/L Tris [pH 8.3] and 25 g/mL bovine serum albumin), 0.8 U of Platinum Taq (Invitrogen), 5 mmol/L MgCl2, and 500 nmol/L each primer. The final concentration of the TaqMan-MGB probe was 100 nmol/L. PCR amplification was performed by use of the following cycling conditions: 2 min at 95°C, followed by 45 cycles of 95°C for 0 s and 60°C for 20 s. Each reaction capillary tube was read in channel 1 (F1) at a gain setting of 15, with fluorescence read at the end of each 60°C step and data being analyzed by use of LightCycler data-analysis software (version 3.5.3).
ELISA for vaccinia-specific rhesus IgG and IgM.
ELISA for vaccinia antibodies was performed as described elsewhere [12], with modifications. Immulon 1B 96-well microtiter plates (Thermo Labsystems) were coated with a suspension of Trioxsalen-treated, UV-inactivated vaccinia virus (NYCBH strain) at a plaque-forming unit equivalent of 106 pfu/well and stored at 4°C overnight. Plates were blocked with PBS that contained 10% fetal bovine serum (FBS) at 37°C for 2 h. The wells were washed with PBS that contained 0.05% Tween-20 (PBST). Serum samples from rhesus monkeys (obtained before and after vaccination) were added to wells in duplicate and serially diluted in PBST/10% FBS (diluent). The diluent was added to some wells to serve as blanks. Plates were incubated with the diluted serum samples at 37°C for 2 h and washed with PBST. For the detection of rhesus IgG or IgM, a horseradish peroxidase (HRP) conjugate of goat antihuman IgG or IgM (Biosource), diluted to 1 : 50,000, was added to the plates, as appropriate. After 1 h of incubation with the HRP-conjugate antibodies, plates were washed 7 times with PBST. The ABTS/H2O2 (1 : 1) HRP substrate system (Kirkegaard & Perry) was added to the wells. Colored reactions were allowed to develop for 30 min, and the reactions were stopped by the addition of 1% SDS to all wells. The absorbance at 405 nm for all plates was determined on a Versamax microplate reader equipped with SOFTmax PRO software (version 3.1.1; Molecular Devices). The antibody titer is the reciprocal of the highest dilution of samples with a mean absorbance at 405 nm greater than the mean absorbance at 405 nm of nonimmune serum samples and 0.05.
RESULTS
Induction of vaccinia-specific neutralizing antibodies in immunodeficient and immunocompetent macaques.
Because most HIV-1infected individuals worldwide lack access to highly active antiretroviral therapy, we wanted to test the protective effect of immunization with Dryvax alone, or of Dryvax preceded by MVA or NYVAC, in untreated simian immunodeficiency virus (SIV)infected macaques. We used 14 Indian rhesus macaques that had been infected with SIVmac251 for 918 months, had virus in their plasma, and had a progressive decrease in CD4+ cell counts (figure 1A) [13, 14]. Four groups of monkeys with CD4+ cell counts <300 cells/mm3 were immunized with 2 doses of MVA followed by Dryvax (group 1), with 2 doses of NYVAC followed by Dryvax (group 2), with Dryvax only (group 3), or with nothing (group 4) (figure 1B). To examine antibody responses in healthy macaques, we also vaccinated 3 uninfected macaques with Dryvax only (group 5) (figure 1B).
Vaccinia neutralizing-antibody titers were measured in all macaques at the time of or a few weeks after Dryvax vaccination. Two inoculations with MVA elicited significantly higher levels of neutralizing antibodies than 2 inoculations with NYVAC (range, 4372042 for MVA vs. 143263 for NYVAC; P = .029, Wilcoxon rank sum test). However, Dryvax did not boost neutralizing-antibody titers any further in most of these previously immunized macaques (figure 2A). SIV-infected macaques immunized only with Dryvax mounted lower neutralizing-antibody responses to vaccinia (group 3) than healthy, uninfected macaques (group 5) (figure 2A), which suggests that different levels of CD4+ cells affected the ability to respond to a single inoculation of Dryvax. However, the fact that the MVA-immunized macaques in group 1 were able to achieve levels of neutralizing antibodies that were comparable to those in the macaques in group 5 indicates that the SIV-infected macaques were still capable of generating a strong neutralizing-antibody response.
As in our earlier study [5], we used imaging to monitor the progression of Dryvax-induced skin lesions in macaques immunized with MVA or NYVAC before vaccination with Dryvax or with Dryvax only. Immunization with these highly attenuated viruses produced no adverse effects, as has also been demonstrated elsewhere [5, 15, 16]. Dryvax lesions were smallest and healed most quickly in MVA-immunized macaques, which also developed higher neutralizing-antibody titers than the NYVAC-immunized macaques (figure 2A and 2B). We assessed this relationship quantitatively by examining the correlation between neutralizing-antibody titers elicited by NYVAC, MVA, or Dryvax; time to maximum lesion size; and time to lesion resolution (data not shown). We found a significant negative correlation between the time to maximum lesion size and neutralizing-antibody titers that was strongest for titers measured before the administration of Dryvax (R = -0.86; P = .0012) and was somewhat weaker 2 and 4 weeks after the administration of Dryvax (R = -0.73; P = .013 and R = -0.64; P = .026, respectively, Spearman rank correlation). These findings confirm and extend our previous observations in a smaller number of immunodeficient macaques [5].
No protection by vaccination of severely immunodeficient macaques against lethal monkeypox virus challenge.
During the course of the study, macaques 770L, Rh22, Rh25, and Rh24 died from AIDS and could not be studied further (figure 1B and table 1). Thus, only 3 macaques each from group 1 (MVA plus Dryvax) and group 2 (NYVAC plus Dryvax), as well as 2 from group 3 and the 2 unvaccinated macaques from group 4, were challenged intravenously with a lethal dose of monkeypox virus 6 months after Dryvax vaccination (table 1). Macaques in group 5 were not challenged.
In samples collected beginning 4 days after the challenge exposure, increasing titers of monkeypox viral DNA were found in the throat of and blood from all the challenged macaques (figure 3). All macaques developed innumerable pox lesions over all body surfaces and the lining of the oropharynx, became severely ill, and were euthanized when they became moribund (days indicated in table 1). There was no significant difference between vaccinated and unvaccinated macaques in the number of DNA genome copies in blood or throat, the course of illness, or the time to death (figure 3 and table 1).
We reasoned that the failure to protect these macaques could be ascribed either to the lack of CD4+ helper function at the time of vaccination or to a decline in protective immune responses during the 6 months since Dryvax vaccination. To distinguish between these possibilities, we designed a second study (figure 4A) in which SIV-infected macaques with CD4+ cell counts of either <300 (macaques Rh41, Rh4, and Rh11; group 6) or 300 (macaques 773L, Rh40, and 783L; group 7) (figure 4B), as well as 2 uninfected healthy control macaques (Rh245 and Rh246; group 8), were immunized with Dryvax and challenged 1 month later with monkeypox virus.
Challenge exposure to monkeypox virus was performed intravenously 1 month after immunization, and viral DNA genomes were detected in the blood of all macaques 3 min after inoculation (table 2). Both uninfected and SIV-infected macaques with CD4+ cell counts 300 cells/mm3 had no viral DNA genomes in blood thereafter and remained healthy (table 2) [7]. In contrast, 2 of the SIV-infected macaques with CD4+ cell counts <300 cells/mm3 had high levels of viral DNA genomes in blood, developed innumerable pocks, and died from disease (table 2). A notable exception was macaque Rh41, which had viral DNA genomes only at day 8 and developed several pocks but managed to survive the challenge. It is possible that the CD4+ cell count at the time of immunization in macaque Rh41 was not representative, because significant fluctuations in the number of CD4+ cells were seen (figure 4B).
Defective immunoglobulin switching in CD4+ celldepleted macaques.
Altogether, these findings suggested that protection might be associated with the development of neutralizing antibodies to vaccinia. However, a discrepancy was that, although an equivalent level of vaccinia neutralizing antibodies was found in immunocompromised macaques immunized with MVA and Dryvax (group 1) and in healthy control macaques immunized with Dryvax (group 8), macaques in group 1 were not protected against monkeypox virus (figures 2A and 4C, tables 1 and 2). We therefore hypothesized the existence of qualitative differences in the vaccinia-specific antibody responses in the immunocompromised macaques, because CD4+ cells are essential for the differentiation and maturation of B cells that secrete antibodies of the appropriate subclass and affinity on antigen encounter [17].
DISCUSSION
The ability of a vaccine to elicit protection in immunocompromised individuals likely depends on its mode of protection and the type of immune deficiency. Here, we demonstrate that a prime-boost regimen that used highly attenuated vaccinia strains (MVA or NYVAC) followed by Dryvax is safe for immunodeficient macaques, as was demonstrated by a previous study [5]. However, no protection was afforded by Dryvax alone or by the combination of Dryvax and highly attenuated poxviruses. The clear-cut relationship between the production of vaccinia-specific IgG and protection against monkeypox virus challenge indicates that the induction of antibodies with high affinity is essential.
IgM constitutes the initial humoral response to most antigens [17]. However, IgM antibodies differ from IgG by having a lower affinity for antigens, a shorter plasma half-life, and a limited ability to diffuse into tissues. It is therefore possible that, in this macaque model of human smallpox, once monkeypox virus has localized to tissues, IgM antibodies may not be effective in blocking viral spread from cell to cell and preventing disease development. Interestingly, our finding that the neutralizing-antibody titer, which was mainly constituted of IgM, was inversely correlated with the size of Dryvax-induced lesions supports the notion that, although the local production of IgM may be sufficient to protect against the uncontrolled enlargement of skin lesions, it is inadequate to protect against systemic monkeypox virus. Human smallpox infection begins when a low dose of airborne virus enters the respiratory tract, followed by spread to lymphoid tissues and the development of viremia that distributes the virus to the skin and mucous membranes [1, 8, 18]. The model that we used simulates the latter part of this process by injecting monkeypox virus directly into the bloodstream [7, 8].
The importance of vaccinia neutralizing antibodies is also supported by our finding that the depletion of B cells precluded protection against monkeypox virus challenge in macaques, whereas the depletion of CD8+ cells before and at time of challenge had no effect (Y. Edghill-Smith, H. Golding, J. Manischewitz, L. R. King, D. Scott, M. Bray, A. Nalca, J. W. Hooper, C. A. Whitehouse, J. Schmitz, K. A. Reimann, and G. Franchini, unpublished data). Indeed, in that study, the passive administration of human vacciniaspecific neutralizing antibodies alone was sufficient to protect macaques against the same lethal monkeypox virus challenge. Thus, the induction of protective immune responses in CD4+ celldeficient individuals will necessitate the development of strategies to restore the appropriate immunoglobulin isotype switching. Such methods might include the use of adjuvants, as well as of cytokines that might boost both native and adaptive immunity. Activation of the Toll 9 receptor by the proper CpG, the CD40 ligand, interleukin-12, or granulocyte-macrophage colony-stimulating factor in conjunction with highly attenuated poxvirus-, DNA-, or protein-based vaccines might help in this endeavor. Vaccine approaches that bypass the need for CD4+ helper cells in immunodeficient individuals will also be instrumental in the development of vaccines to protect against some of the opportunistic infections that occur in HIV-1infected individuals.
Acknowledgments
We thank Jay A. Berzofsky and Igor Belyakov, for helpful discussion; Peter Jahrling, for providing the monkeypox virus stock; Jim Tartaglia, for NYVAC; Bernard Moss, Linda Wyatt, and Patricia Earl, for preparation of the modified vaccinia virus Ankara; Phillip D. Markham, Sharon Orndorff, Jim Treece, and John Parrish, for assistance with some of the macaques; Steven J. Snodgrass, for editorial assistance; and James McNally and Tatiana Karpova, for imaging.
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