In Vitro and In Vivo Fitness of Respiratory Syncytial Virus Monoclonal Antibody Escape Mutants

Abstract

Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.

Human respiratory syncytial virus (RSV) is a leading cause of serious lower respiratory tract infections among infants and young children. RSV infections are estimated to annually cause 0.5 million deaths among children in developing countries and as many as 126,300 hospitalizations among U.S. infants (39, 40). Reinfections are the norm and occur throughout life, and RSV may also cause pneumonia in young adults and the elderly (8, 15, 20). Children who are born prematurely and those with congenital heart disease are at increased risk for severe disease due to RSV (17, 28). Infections in immunocompromised patients may be life threatening (13). No vaccine is available for the prevention of infections due to RSV; however, passive prophylactic immunization with polyclonal or monoclonal antibodies (MAbs) reduces hospitalization rates in high-risk children (23, 36). The monoclonal antibody palivizumab (PZ) is a humanized murine monoclonal antibody that neutralizes RSV through interaction with the RSV fusion (F) glycoprotein. It is the first and, at present, only monoclonal antibody in commercial use against an infectious disease.

RSV is an RNA virus with nonsegmented negative-strand genome and belongs to the Paramyxoviridae family in the order Mononegavirales. The F protein is an important target of both humoral and cellular immune responses. It is a type I transmembrane protein which after synthesis and modification by the addition of N-linked sugars is cleaved into two subunits, F1 and F2, that are linked by disulfide bonds (5).

The quasispecies nature of RNA viruses allows rapid emergence of viruses with a selective advantage (7). Variant RSVs have been selected by growth of virus in the presence of antibody, including mutant selection by the murine antibody parent of PZ (2). We previously described the selection in both cell culture and cotton rats of RSVs resistant to PZ; all had mutations in the F protein (46-48). Fitness for replication might be an important determinant of the capacity of mutant viruses to spread (7). Here we report the use of competitive replication to assess fitness differences between the parent and antibody escape mutant RSV.

MATERIALS AND METHODS

Viruses.

RSV was propagated in HEp-2 cells. The mutant virus resistant to PZ, MP4, was selected as previously described by bulk passage of A2 RSV-infected cell medium through progressively increasing concentrations of PZ (47). At the fifth passage, there was abundant cytopathic effect (CPE) in the presence of 40 μg/ml PZ; this was considered resistant to PZ. A single plaque pick was chosen for further characterization (MP4). The selection process for F212 has also been described and differed from that of MP4 in that, after initial neutralization in the presence of complement, individual plaques were taken and grown in the presence of 40 μg/ml PZ. After 10 passages, CPE was present but was not extensive. Two rounds of plaque purification were performed, and a single plaque was designated F212 (46).

Competitive replication in cell culture.

Mixtures of viruses were inoculated onto HEp-2 cells in 35-mm-diameter wells and cultivated in the absence of PZ. Mutant virus F212 was expected to be less fit in HEp-2 cells as it previously showed impaired growth capacity; it was tested at a 1:1 ratio with A2 virus. F212 (2.5 × 10⁵ PFU) and A2 (2.5 × 10⁵PFU) viruses were mixed and inoculated onto 8 × 10⁵ HEp-2 cells (multiplicity of infection [MOI] = 0.6). MP4 had similar growth kinetics to the parent A2 virus, and a preliminary study suggested MP4 was more fit than A2 (not shown). Therefore, maintaining a cumulative MOI of ∼0.6, MP4 virus was tested with A2 virus at A2/MP4 ratios of 1:1, 10:1, 100:1, and 1,000:1. When viral CPE was abundant, 10 to 20 μl of viral medium was passaged onto fresh HEp-2 cells; this continued for 35 passages. Five hundred microliters of viral media was harvested at each passage and stored at −80°C for viral titer determination. RNA was extracted from infected HEp-2 cells every passage before passage from passages 1 to 10 and subsequently at every 5 passages. A2, MP4, and F212 viruses were passed alone for 35 times as controls.

Differential plaque assay to quantitate wild-type and mutant viruses.

To differentiate mutant from wild-type viruses in the mixed populations, differential plaque assays were performed on Vero cells with or without PZ (4 μg/ml) in the agarose overlay (21). RSV A2 virus was susceptible to PZ and produced no plaques in the presence of PZ. MP4 and F212 formed a similar number of plaques in the presence or absence of PZ. Because the F212 plaques were somewhat smaller in the presence of PZ, the plates were incubated for 5 days after infection, as compared to the 4 days allowed for the other viruses. Antibody-mediated detection of viral plaque assays was performed with a broadly reactive RSV G protein MAb (L9; a generous gift from Ed Walsh, Rochester, NY) for detection of both parent and mutant viruses.

F gene RT-PCR, nucleotide sequence analysis, and determination of relative amount of mixed viruses.

Infected HEp-2 cells provided viral RNA for extraction and reverse transcription-PCR (RT-PCR) amplification. The primer set F622For (5′-GTTACCTATTGTGAACAAGC-3′) and F1225Rev (5′-GCTGCTTACATCTGTTTTTG-3′), spanning the F gene from nucleotides 622 to 1225, yielded a 603-bp fragment after amplification under conditions of 50°C for 45 min, 95°C for 15 min, 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min for 40 cycles followed by 72°C for 5 min. The mutations of MP4 (828 A→T) and F212 (816 A→T) were contained in this fragment. Bulk PCR products were gel purified (QIA Quick Gel extraction kit; QIAGEN, Valencia, CA). Sequencing was done on an automated sequencing machine (ABI model 3100; Applied Biosystems, Inc., Foster City, CA) at the UAB CFAR DNA sequencing core facility using primer F622For.

Visual inspection of the electropherograms and calculation of the relative proportions of the parent and mutant bases were used to assess the viral mixtures. These techniques have been applied previously to assess the relative amounts of viruses present in mixed viral populations (25, 29, 33, 43). We first analyzed the results from experiments that began with predetermined amounts of input DNA. RT-PCR products from A2, MP4, or F212 were quantified by UV spectrophotometry and adjusted to the same concentration. PCR products of A2 and MP4 or A2 and F212 were mixed at ratios of 0/100, 10/90, 25/75, 50/50, 75/25, 90/10, and 100/0 and sequenced. For the A2/F212 mixture, at position 816 in the F gene, base A represented wild-type A2 virus and base T represented the F212 virus. The height of A or T at position 816 was measured using Edit View 1.0.1 (Applied Biosystems, Inc.), and the F212 proportion was obtained by dividing the height of T by the sum of the A and T heights. For the A2/MP4 mixture, nucleotide 828 was assessed for A (A2) and T (MP4) and analyzed by the same method. After this initial assessment of the assay, the same technique was used to assess the results of RT-PCR amplification of the passaged viruses and viral mixtures.

Restriction analysis of F gene PCR products.

A nucleotide change from A to T at 816 in the F gene introduced a restriction site for MfeI in the F212 virus F gene that was not present in the parent A2 virus (A2 nucleotides 813 to 818, CAAATG; F212 nucleotides 813 to 818, CAATTG). RT-PCR products were gel purified and incubated with MfeI at 37°C for 4 h. Digested PCR products were run on 2% agarose gel (4, 42).

Animal model.

Cotton rats (Sigmodon hispidus) obtained commercially (Harlan, Indianapolis, Ind.) were treated with intraperitoneal injections of cyclophosphamide (CY) at 50 mg/kg of body weight three times per week for 3 weeks prior to viral infection (24, 48). CY treatment was continued until the end of study. Cotton rats were infected intranasally with 5 × 10⁵ PFU virus under ketamine-xylazine anesthesia. Group A received A2, group B received MP4, and groups C and D received a 1:1 mixture of A2 and MP4. Groups A, B, and D were sacrificed at 4 weeks after infection, and group C was sacrificed at 4 days after infection. Lungs were harvested for RNA extraction and viral titer assessment by differential plaque assay. Viral RNA was extracted from infected ground lung tissue with Trizol reagent (Invitrogen, Carlsbad, CA), as described previously (47). The region from nucleotides 622 to 1225 in the RSV F gene was amplified as described above and cloned into pCR2.1-TOPO (Invitrogen) as previously described (48). The nucleotide sequences of the insert cDNAs were determined, and the proportion of different genotypes in each population was calculated based on the total number of clones sequenced.

RESULTS

Competitive replication viruses.

Two palvizumab escape mutant viruses, F212 and MP4, were selected previously. The mutant F212 grew to an ∼1-log lower titer than the parent A2 virus in cell culture and in cotton rat lungs. F212 was partially resistant to PZ neutralization in cell culture but was completely eliminated by PZ prophylaxis in cotton rats. F212 had a single nucleotide substitution (816 A to T) resulting in an amino acid change (268 Asn to Ile) (46). MP4 was found to grow as well as A2 virus in cell culture and in cotton rat lungs and was resistant to PZ in cell culture and when administered prophylactically to cotton rats. MP4 had a single nucleotide substitution (828 A to T) resulting in an amino acid change (272 Lys to Met) (47). F212 was thought likely to be less fit than the A2 virus based on its growth characteristics. Because MP4 grew as well as A2, it had no obvious disadvantage relative to A2. In order to assess relative fitness by competitive replication, F212 and A2 or MP4 and A2 were combined and the mixture was used to infect HEp-2 cells. Sequential passages were performed up to passage 35. The proportions of A2, F212, and MP4 present were determined at selected intervals by differential plaque assay, RT-PCR restriction fragment analysis (for F212), and nucleotide sequence determination.

Mixed virus populations assessed by differential plaque assay.

The proportion of F212 virus was nearly 50% at passage 1 in the mixed population, as expected with a 1:1 ratio of infectious virus for the A2 and F212 viruses (Fig. 1A). The F212 proportion continuously decreased until it was undetectable by passage 10. F212 was not detected in subsequent passages, assessed until passage 35. This result was in agreement with the earlier observation that F212 grew to lower titers than the A2 virus in cell culture and in cotton rats (46).

FIG. 1.

Differential plaque assay determination of mutant virus proportions during competitive replication with parent A2 virus. (A) Equal amounts of the parent A2 and the mutant F212 or (B) different initial A2/MP4 ratios (1:1, 10:1, 100:1, and 1,000:1) of the parent A2 and the mutant MP4 were mixed and used to infect HEp-2 cells. Infected-cell medium was passaged to new cells and at the passage numbers shown (x axis) tested by differential plaque assay to determine the proportions of mutant virus present. Duplicate plaque assays were performed on Vero cells, with and without PZ (4 μg/ml) in the agar overlay. The viral titer from the PZ overlay plate represented mutant virus that was resistant to PZ; the viral titer from the plain overlay plate represented both mutant and wild-type A2 virus. The proportion of mutant virus in the mixed population (y axis) was obtained by dividing the viral titer in the PZ plate by that in the no-PZ plate.

The A2 and MP4 viruses were mixed at ratios of 1:1, 10:1, 100:1, and 1,000:1 and passaged 35 times. The relative amount of each virus in selected passes was assessed by differential plaque assay (Fig. 1B). The MP4 virus became dominant in mixtures with input ratios of 1:1, 10:1, and 100:1. This was particularly rapid for the 1:1 mixture, with MP4 already 84% of the mixture at the first passage. For the 10:1 and 100:1 mixtures, A2 was predominant at passages 1 and 5 (≤20% MP4), but by passage 10, MP4 was nearly 60% of the population. The 10:1 mixture was nearly 90% MP4 at passage 15, but at passage 20, the amount of MP4 amount dropped to nearly 15% and then returned to the earlier pattern with over 90% MP4 at passage 25 and with undetectable A2 in later passages. The alteration in the pattern of fitness change at passage 20 for the 10:1 mix was confirmed by analysis of passages 16, 17, 18, and 19. These confirmed a gradual decrease in the proportion of MP4 virus, as compared to the results for passage 15. The 100:1 mixture had little or no A2 found from passage 15 onward. The 1,000:1 ratio differed, and no MP4 was detected until the last passage tested, and even then it was only 2%.

A2/F212 mixed infection assessed by restriction fragment analysis.

The nucleotide substitution in the F212 F gene serendipitously introduced an MfeI restriction site not found in the A2 virus F gene, allowing assessment of the presence of the F212 mutation by restriction fragment analysis. RT-PCR products from dual-infection passages 1 to 5 and passage 10 were digested by MfeI (Fig. 2). PCR products from F212 were digested into 401-bp and 202-bp fragments (F212 control lane), while PCR products from A2 virus remained uncut (A2 control lane). When both viruses were present and three bands were seen, the two lower bands represented F212 and the upper (uncut) band represented A2. Consistent with results by differential plaque assay, the F212 proportion decreased continuously until passage 10, at which F212 was undetectable.

FIG. 2.

Restriction fragment analysis of the dynamics of mutant F212 virus during competitive replication with parent A2 virus. Shown are RT-PCR-amplified DNAs from sequentially passaged A2/F212 mixed viruses (passage numbers shown as p1, p2, etc.) and control A2 and F212 viruses incubated with MfeI and separated by agarose gel electrophoresis. The first lane (standard [std]) is a 100-bp DNA ladder with 200-bp and 600-bp sizes indicated. The A2 virus did not cleave (lane A2), whereas the mutation in the F212 F gene introduced an MfeI site, resulting in two bands after digestion (lane F212). The presence of three bands indicates a mixture of A2 and F212, and a single upper band indicates the presence of A2 without detection of F212.

Assessment of mixed viral populations by analysis of the nucleotide sequence electropherograms.

DNA sequencing has been used to monitor the development of drug resistance mutations in human immunodeficiency virus type 1 and cytomegalovirus (11, 18, 43, 45). These techniques have also quantitated mixed populations in studies of viral fitness (25, 29, 33, 43). We first performed a reconstruction experiment; mixtures of known amounts of the RT-PCR products from the A2 and mutant viruses in various ratios were tested (not shown). The nucleotide sequence electropherograms were visually inspected at the bases of interest, and the relative proportions of the two possible nucleotides were taken from the values in Edit View (PE Applied Biosystems, Inc.). In these experiments, the predetermined input ratios were in close concordance (differing by 4% or less) to the measured proportions and visual inspection of the electropherogram. In this report, we combined a biologic assay (differential plaque assay) with analysis of the sequence electropherogram (visual and calculated) to assess the virus mixtures. As will be seen below, the results were concordant.

Mixed virus populations assessed by nucleotide sequence analysis.

Visual analysis and calculated proportions from the nucleotide sequence electropherogram revealed rapid disappearance of F212 from the mixed population (Fig. 3A). No F212 was detected after passage 10. These results were very similar to those obtained by differential plaque assay from the same passage (Fig. 1). Of note, F212 passaged 35 times alone as a control showed no change in nucleotide sequence at position 816 (results not shown).

FIG. 3.

Dynamics of mutant F212 virus during competitive replication with parent A2 virus assessed by nucleotide sequence analysis. Competitive replication was performed beginning with (A) a 1:1 ratio of the A2 and F212 viruses, or (B) different initial ratios of A2 to MP4 (1:1, 10:1, 100:1, and 1000:1) of the parent A2 and the mutant MP4 were mixed and used to infect HEp-2 cells and passaged sequentially. At the passage numbers shown (x axis), cell lysates were taken for RT-PCR and nucleotide sequence determination. The relative amounts of (A) F gene nucleotide 816 (A from A2 virus and T from mutant F212) or (B) F gene nucleotide position 828 (A from A2 virus and T from mutant MP4) were estimated using Edit View (Applied Biosystems, Inc.), and the proportion of mutant virus in the mixture (T/A + T) is shown (y axis).

The A2 and MP4 viruses were mixed at various ratios and passaged 35 times. The relative amount of each virus in selected passages was assessed by nucleotide sequence analysis, as described above (Fig. 3B). The MP4 virus became dominant in mixes with input ratios of 1:1, 10:1, and 100:1. The 1:1 mixture of A2 to MP4 input virus had no A2 detected after passage 10. The 10:1 ratio mixture also showed rapid diminution in the amount of A2: although A2 unexpectedly dominated at passage 20, A2 subsequently disappeared from the mixture. The 100:1 mixture revealed no A2 after passage 10. However, the 1,000:1 mixture persisted with the A2 virus dominant, with tiny amounts of MP4 detected at passages 5 and 15. The results from sequence analysis paralleled those when competitive replication outcomes were measured by differential plaque assay (Fig. 1). MP4 and A2 controls assessed at passage 35 showed no changes in the site of the MP4 mutation at nucleotide 828.

Multiplicity of infection and competitive replication.

The experiments above demonstrated MP4 to be more fit in competitive replication than the A2 virus. These experiments maintained an MOI of 0.6 as the ratios of the viruses were altered. We speculated the input MOI might influence the outcome of the competitive replication as it has for other viruses (31, 32, 35, 38). Thus, the MOI varied as a 1:1 ratio of the viruses was held constant. The MOIs tested were 0.1, 0.5, 1, and 2. In each instance, the MP4 virus became dominant over the 10 passages as assessed by differential plaque assay (Fig. 4). The results were corroborated by the nucleotide sequence electropherograms (not shown). Thus, varying the MOI of the input virus mixtures by 20-fold did not alter the outcome, and MP4 was more fit than the A2 virus in competitive replication. However, the lowest MOI, 0.1, did have the most rapid and complete shift to MP4 predominance compared to the other MOIs.

FIG. 4.

Dynamics of mutant MP4 virus during competitive replication with parent A2 virus with input mixtures of different multiplicities of infection. Equal amounts of mutant MP4 and A2 virus (by PFU) were mixed and used to infect HEp-2 cells with different initial MOIs (0.1, 0.5, 1, and 2), and infected-cell medium was passaged sequentially to new cells. At the passage numbers shown (x axis), the relative proportion (y axis) of the MP4 virus was determined by differential plaque assay as described in the legend to Fig. 2.

In vivo assessment of fitness.

Cotton rats are permissive to RSV replication and are used as an in vivo model of RSV infection. Because normal cotton rats clear RSV from their lungs within days, immunosuppressed cotton rats were employed here to allow prolonged replication of the viral mixtures and assessment of relative fitness (24). The outcome of the in vivo competitive replication was assessed by differential plaque assay and analysis of cloned cDNAs. Cotton rats were infected with a 1:1 mixture of the A2 and MP4 viruses or the A2 or MP4 viruses alone and were sacrificed at 4 days and 4 weeks after infection. As assessed by differential plaque assay (Table 1), the viruses in the cotton rat lungs infected with the A2/MP4 mixture were predominately of the MP4 phenotype (antibody resistant) at both the 4-day (85%) and 4-week (93%) time points. The lung viral titers from the control animals were determined at 4 weeks after infection. These viruses reflected the antibody resistance phenotype of the input viruses (Table 1). The A2 virus (group A) grew to log₁₀ 5.2 ± 0.5 PFU/g of lung tissue, and the MP4 virus (group B) grew to log₁₀ 4.2 ± 1.6 PFU/g of lung tissue. However, the group B values were lowered by one rat from which no virus was recovered, and the MP4 virus was previously found to replicate as well as the A2 virus in cotton rat lungs (47).

TABLE 1.

Competitive viral replication in immunosuppressed cotton rats^a

Group	n (no. alive at endpoint)	Virus	Time to harvest	% of PZ-resistant virus (no. with no virus detected in lungs)
A	7 (5)	A2	4 wk	0 ± 0
B	7 (5)	MP4	4 wk	106 ± 13 (1)
C	7 (6)	A2/MP4 (1:1)	4 days	85 ± 11 (1)
D	12 (8)	A2/MP4 (1:1)	4 wk	93 ± 8 (0)

^aCyclophosphamide was given to induce immunosuppression as described in Materials and Methods. Infections were performed intranasally with viruses or virus mixtures (A2 and MP4). Deaths occurred in each group, likely due to immunosuppression or complications of anesthesia. Lungs were taken at the times shown, differential plaque assay were performed to determine the amounts of each virus present, and the proportion of antibody-resistant virus was calculated.

Viral RNA extracted from the cotton rat lungs was amplified by RT-PCR, and nucleotide sequences were determined from cloned F gene cDNAs (Table 2). Control animals infected with single viruses were tested at 4 weeks. As expected, the individual input viruses dominated: A2-infected animals were 98% A2, and MP4-infected animals were 92% MP4. The results from the samples from 4 days and 4 weeks postinfection differed among animals infected with the A2 and MP4 virus mixtures. At 4 days, the cloned DNAs were 45% MP4, 12% A2, and 38% F212. At 4 weeks, MP4 was dominant, and the clones were 78% MP4, 10% A2, and 10% F212. Thus, analysis of molecular clones revealed additional complexity of the viral populations as compared to phenotypic testing based on antibody susceptibility (Table 1). However, the results were concordant, and the proportions of MP4 virus exceeded that for A2 at both the 4-day and 4-week time points.

TABLE 2.

Analysis of cloned amplified F gene products from the lungs of infected cotton rats^a

Rat and input virus (time of postinfection harvest)	No. of clones	No. (%) of rats with output virus:
		MP4 (828 A→T)	A2	F212 (816 A→T)	MS412 (827 A→C)	816 A→G, 827 A→C	828 A→C
A2 (4 wk)
A-1	10	0	10 (100)	0	0	0	0
A-2	9	0	9 (100)	0	0	0	0
A-4	10	0	10 (100)	0	0	0	0
A-5	10	0	9 (90)	1 (10)	0	0	0
A-6	10	0	10 (100)	0	0	0	0
Total	49		48/49 (98)	1/49 (2)
MP4 (4 wk)
B-1	10	9 (90)	1 (10)	0	0	0	0
B-2	10	10 (100)	0	0	0	0	0
B-3	10	9 (90)	1	0	0	0	0
B-4	9	7 (78)	1 (11)	1 (11)	0	0	0
B-5	10	10 (100)	0	0	0	0	0
Total	49	45/49 (92)	3/49 (6)	1/49 (2)
A2/MP4 (4 days)
C-1	20	15 (75)	4 (20)	0	1 (5)	0	0
C-2	20	6 (30)	4 (20)	9 (45)	1 (5)	0	0
C-3	19	3 (16)	2 (11)	13 (68)	0	1 (5)	0
C-5	18	1 (6)	0	17 (94)	0	0	0
C-6	18	9 (47)	3 (15.8)	4 (21)	2 (11)	0	0
C-7	17	16 (94)	0	0	0	0	1 (6)
Total	112	50/112 (45)	13/112 (12)	43/112 (38)	4/112 (4)	1/112 (1)	1/112 (1)
A2/MP4 (4 wk)
D-1	19	17 (90)	1 (5)	0	1 (5)	0	0
D-3	18	16 (89)	2 (11)	0	0	0	0
D-5	20	18 (90)	0	2 (10)	0	0	0
D-6	18	18 (100)	0	0	0	0	0
D-7	20	4 (20)	2 (10)	14 (70)	0	0	0
D-8	19	16 (84)	2 (11)	0	1 (5)	0	0
D-10	20	15 (75)	4 (20)	0	1 (5)	0	0
D-12	20	16 (80)	4 (20)	0	0	0	0
Total	154	120/154 (78)	15/154 (10)	16/154 (10)	3/154 (2)

^aImmunosuppressed cotton rats infected with the viruses or mixtures of viruses were harvested at the times indicated. These are the same animals for which viral titer results are shown in Table 1. RNA was extracted from infected cotton rat lungs, RT-PCR products were cloned into a plasmid vector, and nucleotide sequences of the inserted DNA (nucleotides 660 to 1210 in the F gene) were determined. Differences compared to the A2 virus F gene sequence are shown; if the same mutation has been observed in previously described escape mutants, the name of the previous mutant is provided (48). The 828 A→T (virus MP4) and 827 A→C (virus MS412) changes resulted in two different changes at amino acid 272 in the F1 subunit (Lys to Met or Lys to Gln, respectively). The position 828 A→C mutation predicted a coding change in amino acid 272 (Lys to Thr). A mutation at another site, 816 A→T, led to an amino acid substitution from Asn to Ile at position 268 (virus F212) and 816 A→G predicted another 272 coding change (Asn to Ser).

DISCUSSION

RSV is the most commonly identified viral cause of lower respiratory tract infections in infants and young children worldwide. Antigenic variation may contribute to the capacity of RSV to establish infections in the presence of preexisting immunity (34). The existence of RNA viruses as quasispecies or populations of viruses with nonidentical genomes provides opportunities for variant viruses to arise under selective pressure (7). Antibody escape mutants of RSV have been readily selected in cell culture and in cotton rats by growth in the presence of antibody (16, 26, 41, 48). However, antibody escape mutants may be less fit than their parent viruses (10, 21). If mutant viruses are less fit, the advantage conferred by resistance to antibody would be moderated by an associated disadvantage in growth, particularly when not in the presence of the selecting antibody. Escape mutants and their fitness could have important implications for RSV, the only infectious disease for which a monoclonal antibody is in clinical use in humans. PZ is a humanized murine monoclonal antibody to the F protein of RSV. PZ is used prophylactically against RSV in selected high-risk infants and children. In this report, we describe an investigation of the fitness of two mutant RSVs resistant to PZ: one of the mutants (F212) was less fit than the parent virus and the other mutant (MP4) was more fit.

Fitness outcomes were measured by both biologic (differential plaque assay) and molecular techniques (nucleotide sequence determination and, for F212, restriction fragment analysis), and the results of the different assays were congruent. F212 grew less well and, as expected, was less fit in in vitro competitive replication than the parent A2 virus (48). The selection process techniques may have increased the likelihood of a less fit virus arising. A single plaque was taken after the first neutralization by antibody, and after 10 passages, two more plaque picks were done. The initial clonal selection of F212 may have served as a genetic bottleneck with a subsequent founder effect. Among asexual organisms with high mutation rates in which a large proportion of the mutations are deleterious, passage of small populations typically results in a reduction in fitness. The initial plaque pick may have chosen a less fit virus, as suggested by the observation that the virus did not have extensive CPE after this plaque pick. With repeated passages of small populations, there will be progressive diminution of fitness, a process known as Muller's ratchet (9). Thus, the later selection of individual plaques might have also reduced viral fitness.

The assessment of the MP4 virus yielded strikingly different results and revealed MP4 was more fit than the parent virus. Unlike the situation for F212, this result was not predicted by the growth characteristics of MP4 in cell culture and in cotton rats, which were identical to those of the parent virus (47). This mirrors recent work with vesicular stomatitis virus and measles virus, which noted that viruses with indistinguishable growth curves might be very different in fitness (4, 31).

The different fitness outcomes between F212 (less fit) and MP4 (more fit) may be due to their different selection processes. Unlike F212, MP4 underwent bulk passages first and then a single round of plaque purification. The fitness of the quasispecies population from which MP4 was taken was unknown and may have been more or less fit than MP4. In any event, this is a stochastic process, and not all bottlenecks result in diminished fitness (10, 14, 30).

Competitive replication for MP4 was done in various ratios with A2 while holding the MOI the same. At the three lowest ratios, MP4 became the dominant virus in the mixture. This may be compared to the results seen with vesicular stomatitis virus, where fitness values do not change significantly if the MOI is held constant while the population size varies (31, 32). However, because we held both the MOI and the total population size constant, the proportions of each virus in the population varied. During multiple passages of a mixture of two viruses, the more abundant virus has greater chance of an increase in fitness (21). Conversely, the less abundant virus has a greater probability of a decrease in fitness via Muller's ratchet (9). This may explain why the MP4 virus was not able to outcompete the A2 virus when there was a thousand-fold less MP4 than A2 virus in the starting mixture.

Fitness comparisons were also performed in cotton rats for the MP4 and A2 viruses. Immunosuppressed cotton rats were used to allow a more prolonged period of competitive replication (24). Phenotypic characterization of the mixed virus populations at 4 days and 4 weeks postinfection revealed that antibody-resistant viruses predominated over antibody-susceptible viruses (Table 1). Analysis of cloned cDNAs revealed the complexity of these viral populations, as we observed previously when we demonstrated that PZ escape mutants can arise during prolonged RSV replication in the presence of PZ (Table 2) (48). For instance, the lungs of cotton rats infected with the A2/MP4 mixture and harvested at 4 days were 45% MP4, 12% A2, 38% F212, and the remaining 6% was comprised of three different F gene mutations. A2 was not the dominant virus in any individual rat, although in some rats F212 actually was more commonly identified than MP4. At the 4-week harvest point the MP4 virus was clearly dominant in seven of eight rats tested. Thus, by both in vitro and in vivo assessments of competitive replication, the MP4 virus was more fit than the A2 virus.

PZ is the first and thus far only monoclonal antibody used in humans against an infectious disease. Therefore, it is of significant interest as a prototype for monoclonal antibody prophylaxis against an infectious disease. This is particularly true since passively administered antibodies are being considered for use against other infections, including emerging viruses and agents of biodefense. Testing of RSV clinical isolates, including 25 viruses from PZ recipients, found uniform reactivity with PZ (6). There are reasons PZ-resistant viruses might not arise or if they did arise would be unlikely to disseminate in the community. Typically, the normal immune responses of the host will clear the virus, reducing both the opportunity for the selection of variant viruses and for variants to spread. This situation would be altered when PZ is given to an immunocompromised host who experiences a prolonged period of viral replication. However, escape mutants were not observed in one reported case of this nature (12). Also limiting the opportunities for spread is the fact that PZ is given to a select group and only a tiny portion of the population is exposed to it. In addition, the attribute of PZ resistance alone likely does not provide a selective advantage when the virus is passed to a person who is not receiving PZ.

The preceding data are encouraging with regard to the risk of development of PZ resistance. However, the murine monoclonal antibody from which PZ was derived failed to neutralize one RSV clinical isolate, establishing that circulating wild-type viruses may be resistant to PZ (2). As noted above, we demonstrated in cotton rats that prolonged replication of RSV in the presence of PZ was followed by the appearance of PZ-resistant viruses (48). There are also other experimental examples of viral variants arising in actively (influenza virus and foot-and-mouth disease virus) and passively (parvovirus) immunized animals (19, 22, 27). Hepatitis B virus mutants have been found in humans after active immunization and among liver transplant patients passively immunized with hepatitis B virus immunoglobulin (3, 37). Consideration of these facts and of the quasispecies nature of RNA viruses suggests the potential for the development of resistance remains.

The two PZ-resistant viruses described here were found to differ in fitness from the parent virus: one virus was more fit and the other was less fit. The mutant viruses had nucleotide substitutions in the F gene that altered single residues in the deduced amino acid sequence compared to the parent virus. It seems likely these are the changes that confer PZ resistance. Given the multiple passages of these viruses in cell culture, there may be other changes elsewhere in the genome. Potentially any step in the viral life cycle might be altered and result in fitness changes, including virus attachment and entry into the cell, nucleocapsid uncoating, viral gene expression, genome replication, virus particle assembly, and virus release from the cell (1).

Extensive work in the development of RSV vaccine candidates has identified a number of genetic changes that result in attenuation (44). However, viruses of increased fitness have not been previously described. Defining the mechanisms that alter fitness in the mutant viruses reported here could provide data that are useful in the design of RSV vaccines.